RU2769465C2 - Method of producing dna and a kit for binding dna fragments - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: present invention relates to a method of producing linear or circular DNA and a kit for binding DNA fragments used in said method. Linear or circular DNA is obtained in a reaction solution by linking DNA fragments of two or more types to each other in areas, having homologous base sequences, or in regions having complementary base sequences, in one reaction. Regions having homologous base sequences, or regions having complementary base sequences, are present at the end of the DNA fragment or near it and have length of 20 b. s. or more. Reaction of binding DNA fragments of two or more types is carried out at a temperature in range of 25–48 °C. Reaction solution contains a regenerating enzyme for nucleoside triphosphates or deoxynucleotide triphosphates and a substrate thereof.

EFFECT: method enables to efficiently bind DNA of two or more types using recombinase of the RecA family and exonuclease.

16 cl, 29 dwg, 1 tbl, 25 ex

Description

Technical field

[0001] The present invention relates to a method for producing linear or circular DNA by linking two or more types of DNA fragments to each other in regions having homologous base sequences, and to a DNA fragment binding kit used in this method.

This application claims priority of Japanese Patent Application No. 2017-132084, filed July 5, 2017, and Japanese Patent Application No. 2017-231732, filed December 1, 2017, the contents of which are incorporated herein by reference.

State of the art

[0002] This application describes a method for producing linear or circular double-stranded DNA by linking a plurality of linear double-stranded DNA fragments. In this method, longer double-stranded DNA, which is difficult to synthesize by chemical synthesis, can be obtained. Methods for linking linear double-stranded DNA fragments mainly include a ligation method (Patent Document 1) and a Gibson assembly method (Patent Document 2 and Patent Document 3).

[0003] The ligation method is a method in which the binding reaction is carried out using a hybrid enzyme having the function of recognizing the homologous sequence of the terminal 15 bases of each double-stranded DNA fragment and binding them. In particular, first, a homologous region consisting of a sequence of identical bases is attached to the ends of two double-stranded DNA fragments linked by PCR. Two double-stranded DNA fragments, at both ends of which a 15-base homologous region has been attached, are linked by mixing with the hybrid enzyme and incubating.

[0004] On the other hand, in the Gibson assembly method, first, the distal region of the first DNA molecule and the proximal region of the second DNA molecule are cleaved using an enzyme having exonuclease activity. As a result, the corresponding homologous regions (regions with identical sequences that are long enough to specifically hybridize with each other) become single stranded. Then, after their specific annealing and binding, gaps and nicks are subjected to repair to obtain a full-length molecule with associated double-stranded DNA. For example, Patent Document 2 discloses 1.6 kb double-stranded DNA fragments. and 1.4 kb, obtained in single-stranded form by cleavage with T4 DNA polymerase, which has exonuclease activity. The resulting two single-stranded DNAs were linked together in the presence of added RecA, dNTP, and DNA ligase, and then the gap was completed by the polymerase activity of this T4 DNA polymerase to repair nicks, and a 3 kb double-stranded DNA was obtained. .

[0005] In addition, Patent Document 4 describes a method for linking a plurality of linear double-stranded DNA fragments using RecA. This method includes the step of incubating a plurality of linear double-stranded DNA fragments with DNA ligase in the presence of a RecA family recombinase or a protein having recombinant activity to obtain a linear double-stranded DNA in which a plurality of linear double-stranded DNA fragments are connected to each other in series. In this method, using RecA family recombinase enzymes or the like. the binding of both ends of the linear double-stranded DNA fragment is inhibited and the binding of the ends of the linear double-stranded DNA fragments is stimulated.

List of cited documents

patent document

[0006] Patent document 1:. US Patent No. 7575860.

Patent Document 2: US Patent No. 7776532.

Patent Document 3: US Patent No. 8968999.

Patent Document 4: Japanese Unexamined Patent Application First Publication No. 2016-077180.

The essence of the invention

Technical problem

[0007] Using the ligation method and the Gibson assembly method, two linear DNA fragments can be linked to each other without any problem. However, the disadvantage of these methods is that the number of linked fragments increases and the binding efficiency decreases, and therefore the linked molecule of interest cannot be obtained.

[0008] It is an object of the present invention to provide a method for producing linear or circular DNA by linking two or more types of DNA fragments to each other in regions having homologous base sequences, and to obtain a DNA fragment binding kit used in this method.

Remedies for this problem

[0009] As a result of intensive research, the authors of the present invention found that two or more types of DNA can be efficiently linked using a RecA family recombinase and an exonuclease, and on this basis, the present invention was created.

[0010] A method for producing DNA and a kit for binding DNA fragments according to the invention are presented below in [1]-[31].

[1] A method for producing DNA, wherein said method comprises:

obtaining a reaction solution containing DNA fragments of two or more types and a protein having recombinase activity of the RecA family, and

obtaining linear or circular DNA in the reaction solution by linking two or more types of DNA fragments to each other in regions having homologous base sequences or in regions having complementary base sequences.

[2] The DNA production method according to [1], wherein the reaction solution also contains an exonuclease.

[3] The DNA production method according to [2], wherein the exonuclease is 3'→5'-exonuclease.

[4] The DNA production method according to [1], wherein the reaction solution also contains a 3'→5'-exonuclease specific for linear double-stranded DNA.

[5] The DNA production method according to [1], wherein the reaction solution also contains a 3'→5'-exonuclease specific for linear double-stranded DNA and a 3'→5'-exonuclease specific for single-stranded DNA.

[6] The method for producing DNA according to any one of [1] to [5], wherein the reaction solution contains a regenerating enzyme for nucleoside triphosphates or deoxynucleotide triphosphates and a substrate thereof.

[7] The method of obtaining DNA in accordance with [6], where the regenerating enzyme is creatine kinase, and its substrate is creatine phosphate; the regenerating enzyme is pyruvate kinase, and its substrate is phosphoenol pyruvate; the regenerating enzyme is acetate kinase, and its substrate is acetyl phosphate; the regenerating enzyme is polyphosphate kinase and its substrate is polyphosphate; or the regenerating enzyme is a nucleoside diphosphate kinase and its substrate is a nucleoside triphosphate.

[8] The method for producing DNA according to any one of [1] to [7], wherein the reaction solution at the beginning of the binding reaction of two or more types of DNA fragments has a magnesium ion source concentration of 0.5 to 15 mM, and a concentration of nucleoside triphosphate or deoxynucleotide triphosphate 1-1000 μM.

[9] The method for producing DNA according to any one of [1] to [8], wherein the binding reaction of DNA fragments of two or more types is carried out at a temperature in the range of 25-48°C.

[10] The DNA production method according to any one of [1] to [9], wherein linear or circular DNA is obtained by linking 7 or more DNA fragments.

[11] The method for producing DNA according to any one of [1] to [10], wherein the reaction solution contains one or more substances selected from the group consisting of tetramethylammonium chloride and dimethyl sulfoxide.

[12] The method for producing DNA according to any one of [1] to [11], wherein the reaction solution contains one or more substances selected from the group consisting of polyethylene glycol, an alkali metal ion source, and dithiothreitol.

[13] The method for producing DNA according to any one of [1] to [12], wherein the protein having recombinase activity of the RecA family is uvsX, and the reaction solution also contains uvsY.

[14] The method for producing DNA according to any one of [1] to [13], wherein regions having homologous base sequences or regions having complementary base sequences are present at or near the end of a DNA fragment.

[15] The method of obtaining DNA in accordance with [14], where areas having homologous base sequences, or areas having complementary base sequences, have a length of 10 p. up to 500 bp

[16] The method for producing DNA according to any one of [1] to [15], wherein the reaction solution at the beginning of the binding reaction of two or more types of DNA fragments contains two or more types of DNA fragments at the same molar concentration.

[17] A method for producing DNA according to any one of [1] to [16], also comprising repairing gaps and nicks in the resulting linear or circular DNA using gap repair enzymes.

[18] The method for producing DNA according to [17], also including heat treatment of the resulting linear or circular DNA at 50-70°C, followed by rapid cooling to a temperature of 10°C or less, and then repair of gaps and nicks using repair enzymes gaps.

[19] A method for producing DNA according to [17] or [18], also including amplification of linear or circular double-stranded DNA with gap and nick repair.

[20] The DNA production method according to any one of [1] to [16], wherein the DNA obtained by binding is linear, and PCR is carried out directly using linear DNA as a template.

[21] The method for producing DNA according to any one of [1] to [16], wherein the DNA obtained by binding is a circular DNA containing an origin of replication sequence capable of binding to an enzyme having DnaA activity, and

form a reaction mixture containing cDNA, a first group of enzymes that catalyze the replication of cDNA; the second group of enzymes that catalyze the binding reaction of Okazaki fragments and synthesize two sister circular DNA, consisting of catenan; a third group of enzymes that catalyze the separation of two sister circular DNAs, and dNTPs.

[22] The method for producing DNA according to [21], also including preliminary heat treatment of the obtained DNA at 50-70°C, followed by rapid cooling to a temperature of 10°C or less, and then obtaining a reaction mixture.

[23] The method for producing DNA according to any one of [1] to [16], also including introducing the resulting linear or circular DNA into a microorganism, and amplifying double-stranded DNA with gap and nick repair.

[24] A kit for binding DNA fragments, comprising: a protein having recombinase activity of the RecA family, where the specified kit is used to obtain linear or circular DNA by linking two or more types of DNA fragments to each other in areas having homologous base sequences, or in regions that have complementary base sequences.

[25] Kit for binding DNA fragments according to [24], also including exonuclease.

[26] Kit for binding DNA fragments in accordance with [25], where the exonuclease is 3'→5'-exonuclease.

[27] A kit for binding DNA fragments according to [24], also containing an exonuclease specific for linear double-stranded DNA.

[28] A kit for binding DNA fragments according to [24], also containing an exonuclease specific for linear double-stranded DNA and a 3'→5'-exonuclease specific for single-stranded DNA.

[29] A kit for binding DNA fragments according to any one of [24]-[28], which also contains a regenerating enzyme for nucleoside triphosphates or deoxynucleotide triphosphates and its substrate.

[30] The kit for binding DNA fragments according to any of [24]-[29], also containing one or more substances selected from the group consisting of tetramethylammonium chloride and dimethyl sulfoxide.

[31] A kit for binding DNA fragments according to any of [24]-[30], also containing one or more substances selected from the group consisting of nucleoside triphosphate, deoxynucleotide triphosphate, magnesium ion source, alkali metal ion source , polyethylene glycol, dithiothreitol and buffer.

Advantageous Effects of the Invention

[0011] Using the DNA production method of the invention, multiple DNA fragments can be efficiently linked, resulting in linear or circular DNA.

By using a DNA binding kit, the method of obtaining DNA can be simplified and DNA fragments can be efficiently linked to each other.

Brief description of the drawings

[0012] FIG. 1 is a diagram schematically illustrating an embodiment in which linear double-stranded DNA fragments are linked to each other in accordance with the principle of the method for producing DNA according to the invention.

In FIG. 2 is a color image of the bands separated by agarose gel electrophoresis for the reaction solution obtained by the 7 fragment binding reaction in Example 1.

In FIG. 3 is a color image of the bands separated by agarose gel electrophoresis for the reaction solution obtained by the 5-fragment binding reaction in Example 2.

In FIG. 4 is a color image of bands separated by agarose gel electrophoresis for the reaction solution obtained by the 5-fragment coupling reaction in Example 3.

In FIG. 5 is a color image of the bands separated by agarose gel electrophoresis for the reaction solution obtained by the 7 fragment binding reaction in Example 4.

In FIG. 6 is a color image of the bands separated by agarose gel electrophoresis for the reaction solution obtained by the 5-fragment binding reaction in Example 5.

In FIG. 7 is a color image of the bands separated by agarose gel electrophoresis for the reaction solution obtained by the 7 fragment binding reaction in Example 5.

In FIG. 8 is a color image of the bands separated by agarose gel electrophoresis for the reaction solution obtained by the 7 fragment binding reaction in Example 6.

In FIG. 9 is a color image of the bands separated by agarose gel electrophoresis for the reaction solution obtained by the 7 fragment binding reaction in Example 7.

In FIG. 10 is a color image of the bands separated by agarose gel electrophoresis for the reaction solution obtained by the coupling reaction of 20-49 fragments in Example 8.

In FIG. 11 is a color image of the bands separated by agarose gel electrophoresis for the reaction solution obtained by the 25-fragment binding reaction in Example 9.

In FIG. 12 shows (a) color image of the bands separated by agarose gel electrophoresis for the reaction solution obtained by the coupling reaction of 21 or 26 fragments, and (b) color image of the bands separated by agarose gel electrophoresis for the reaction mixture obtained by additional RCR amplification after the coupling reaction in Example 10.

In FIG. 13 shows (a) color image of the bands separated by agarose gel electrophoresis for the reaction solution obtained by the 26 fragment coupling reaction, and (b) color image of the bands separated by agarose gel electrophoresis for the reaction mixture obtained by additional RCR -amplification after the binding reaction in Example 11.

In FIG. 14 shows (a) color image of the bands separated by agarose gel electrophoresis for the reaction solution obtained by the 26 or 36 fragment coupling reaction, and (b) color image of the bands separated by agarose gel electrophoresis for the reaction mixture obtained by additional RCR amplification after the coupling reaction in Example 12.

In FIG. 15 shows (a) color image of the bands separated by agarose gel electrophoresis for the reaction solution obtained by the 26-fragment binding reaction by the binding method (RA) according to the invention and the NEB method, and (b) color image of the bands separated by electrophoresis in agarose gel for the reaction mixture obtained by additional RCR amplification after the coupling reaction in Example 13.

In FIG. 16 is a color image of bands separated by agarose gel electrophoresis for a reaction mixture prepared by coupling an Xba I digest of E. coli genomic DNA to a fragment containing oriC to generate cDNA followed by RCR amplification in Example 14.

In FIG. 17 is a color image of the bands separated by agarose gel electrophoresis for the reaction solution obtained by the coupling reaction of 10 fragments in the reaction solution containing the ATP regeneration system consisting of creatine kinase (CK) and creatine phosphate (CP) in Example 15.

In FIG. 18 is a color image of the bands separated by agarose gel electrophoresis for the reaction solution obtained by the coupling reaction of 10 fragments in the reaction solution containing the ATP regeneration system consisting of creatine kinase and creatine phosphate in Example 16.

In FIG. 19 is a color image of bands separated by agarose gel electrophoresis for a reaction solution obtained by coupling reaction of 36 fragments in a reaction solution containing an ATP regeneration system consisting of pyruvate kinase (PK) and phosphoenol pyruvate (PEP) in Example 17.

In FIG. 20 is a color image of the bands separated by agarose gel electrophoresis for the reaction solution obtained by the coupling reaction of 10 fragments in the reaction solution containing the ATP regeneration system consisting of polyphosphate kinase (PPK) and polyphosphate (PP) in Example 18.

In FIG. 21 is a color image of the bands separated by agarose gel electrophoresis for the reaction solution obtained by the coupling reaction of 10 fragments in the reaction solution containing exonuclease III and exonuclease I in Example 19.

In FIG. 22 shows (a) color image of the bands separated by agarose gel electrophoresis for the reaction solution obtained by the 36 fragment coupling reaction and (b) color image of the bands separated by agarose gel electrophoresis for the reaction mixture obtained by additional RCR- amplification after the binding reaction in Example 20.

In FIG. 23 shows (a) color image of the bands separated by agarose gel electrophoresis for the reaction solution obtained by the coupling reaction of 50 or 36 fragments and (b) color image of the bands separated by agarose gel electrophoresis for the reaction mixture obtained by additional RCR amplifications after the coupling reaction in Example 21.

In FIG. 24 is a color image of bands separated by agarose gel electrophoresis of the DNA extracted from the transformant in Example 21. The transformant was obtained by introducing DNA into E. coli in a reaction solution obtained by additional RCR amplification after a 50 fragment binding reaction.

In FIG. 25 is a color image of the bands separated by agarose gel electrophoresis for enzymatic digestion of the amplified product obtained by RCR amplification of the DNA extracted from the obtained transformant in Example 21.

In FIG. 26 is a color image of the bands separated by agarose gel electrophoresis for the reaction solution obtained by the 2-fragment binding reaction in Example 22.

In FIG. 27 is a color image of the bands separated by agarose gel electrophoresis for the reaction solution obtained by the coupling reaction of 10 fragments in the reaction solution containing exonuclease III, exonuclease I and exonuclease T in Example 23.

In FIG. 28 is a color image of bands separated by agarose gel electrophoresis for a reaction solution prepared by coupling reaction of 10 fragments in a reaction solution containing UvsX, the RecA bacteriophage homologue in Example 24.

In FIG. 29 is a color image of the bands separated by agarose gel electrophoresis for the reaction solution obtained by the coupling reaction of 10 fragments in the reaction solution containing UvsX and UvsY in Example 25.

Description of embodiments of the invention

[0013] <Method for obtaining DNA>

In the method for producing DNA according to the invention, DNA fragments with regions having base sequences that are homologous to each other (hereinafter, sometimes simply referred to as "homologous regions"), or with regions having base sequences that are complementary to each other (hereinafter, sometimes simply referred to as "complementary regions" ), bind to each other in homologous regions or in complementary regions to obtain linear or circular DNA. Since the method for producing DNA according to the invention is carried out by a binding reaction in the presence of a protein having recombinase activity of the RecA family, the binding efficiency of this method is excellent.

[0014] In the present invention and in the present description, the phrase "base sequences are homologous" means that "the base sequences are identical", and the phrase "nucleotide sequences are complementary" means that "the base sequences are complementary to each other".

[0015] In particular, in the method for producing DNA according to the invention, a reaction solution is prepared containing two or more types of DNA fragments, and a protein having recombinase activity of the RecA family (hereinafter, sometimes referred to as "RecA family recombinase protein"), and DNA fragments of two or more types bind to each other in homologous regions or in complementary regions in the reaction solution. This method allows you to get a linear or circular DNA. Further, linear or circular DNA in which two or more types of DNA fragments are linked may be referred to as a "linked molecule".

[0016] In the method for producing DNA according to the invention, the associated DNA fragment may be a linear double-stranded DNA fragment or a single-stranded DNA fragment. That is, linear double-stranded DNA fragments can be linked to each other; a linear double-stranded DNA fragment and a single-stranded DNA fragment can be linked to each other; or single-stranded DNA fragments can be linked to each other. Linear double-stranded DNA fragments of one or more types and single-stranded DNA fragments of one or more types may be linked. When linking linear double-stranded DNA fragments, or when linking a linear double-stranded DNA fragment and a single-stranded DNA fragment, both of these fragments are linked to each other in a homologous region. When linking linear single-stranded DNA fragments, both of these fragments are associated with each other in the complementary region.

[0017] If the associated DNA fragment of at least one type is a linear double-stranded DNA fragment in the DNA production method of the present invention, then the reaction solution also contains an exonuclease.

[0018] FIG. 1 is a diagram schematically illustrating an embodiment in which linear double-stranded DNA fragments are linked to each other in accordance with the principle of the method for producing DNA according to the invention. First, 3'→5'-exonuclease 2 acts on linear double-stranded DNA fragment 1a and linear double-stranded DNA fragment 1b, which have a homologous H region, causing this homologous H region to become single stranded. The RecA 3 family recombinase protein acts on the homologous H region in single stranded form, and the homologous H regions complementary to each other are subjected to hybridization, whereby the linear double stranded DNA fragment 1a and the linear double stranded DNA fragment 1b become single stranded. As shown on the right in FIG. 1, cleavage by 3'→5'-exonuclease 2 can only be performed on one linear double stranded DNA fragment 1a or on one linear double stranded DNA fragment 1b. Thus, for example, the homologous region H of linear double stranded DNA fragment 1a in single stranded form acts on the homologous region H of linear double stranded DNA fragment 1b in double stranded form in the presence of the RecA 3 family recombinase protein, and both of these fragments are linked.

[0019] When linking linear double-stranded DNA fragments, or when linking a linear double-stranded DNA fragment and a single-stranded DNA fragment, in the method for producing DNA according to the invention, the double-stranded DNA fragment is cleaved with an exonuclease to form a homologous region in single-stranded form, and then a binding reaction is carried out in the presence of a recombinase protein Reca family. For this reason, the method for producing DNA according to the invention is excellent in terms of binding efficiency and allows the binding of a plurality of linear double-stranded DNA fragments in one reaction, which is difficult to achieve by standard methods.

[0020] When linking linear single-stranded DNA fragments in the method of obtaining DNA according to the invention, the RecA family recombinase protein rapidly forms strands on each single-stranded DNA fragment, which leads to inhibition of exonuclease cleavage. Thereafter, homologous H regions that are complementary to each other hybridize with each other under the action of the RecA family recombinase protein, and both single-stranded DNA fragments are linked.

[0021] In the method for producing DNA according to the invention, the number of linked DNA fragments is preferably 5 (5 fragments) or more, more preferably 7 (7 fragments) or more, even more preferably 10 (10 fragments) or more, and may be equal to 20 (20 fragments) or more. The upper limit of the number of linked DNA fragments in the method of obtaining DNA according to the invention is not particularly limited. So, for example, up to 100 fragments can be linked. In the method for producing DNA according to the invention, for example, about 50 linear double-stranded DNA fragments can be linked by optimizing reaction conditions and the like. In the method for producing DNA according to the invention, the DNA fragments may be linked to each other and may be from different species. This method also allows DNA fragments containing two or more DNA fragments of the same type to be linked.

[0022] Each associated DNA fragment of two or more types according to the invention includes a homologous region or a complementary region for binding to at least one of the other DNA fragments. In the method for producing DNA according to the invention, when linking linear double-stranded DNA fragments, or when linking a linear double-stranded DNA fragment and a single-stranded DNA fragment, first one strand of the double-stranded DNA fragment is cleaved with an exonuclease to obtain a homologous region in single-stranded form. For this reason, the homologous region is preferably present at the end of a linear double-stranded DNA fragment. A homologous region may be present near this end. Thus, for example, the base on the terminal side of the linear double-stranded DNA fragment at the end of the homologous region is preferably within 300 bases of the end, more preferably within 100 bases, even more preferably within 30 bases, and even more preferably within within 10 bases. On the other hand, upon binding of linear single-stranded DNA fragments, exonuclease cleavage is inhibited by the action of strands of RecA family recombinase proteins. Thus, a complementary region can be present in any part of a single-stranded DNA fragment.

[0023] The base sequences of homologous regions or complementary regions may be the same base sequence in all linked DNA fragments. Preferably, the base sequences of the homologous regions in the linked DNA fragments differ in each type of DNA fragment in order of location. So, for example, to link double-stranded DNA fragment A, double-stranded DNA fragment B, and double-stranded DNA fragment C in this order, homologous region a is placed at the downstream end of double-stranded DNA fragment A and at the upstream end of double-stranded DNA fragment B, and homologous region b is placed at the downstream end of double-stranded DNA fragment B and to the upstream end of double-stranded DNA fragment C. Double-stranded DNA fragment A and double-stranded DNA fragment B are linked to each other in the homologous region a. Double-stranded DNA fragment B and double-stranded DNA fragment C are linked to each other in a homologous region b. This makes it possible to obtain a linear DNA in which double-stranded DNA fragment A, double-stranded DNA fragment B, and double-stranded DNA fragment C are linked to each other in this order. In this case, homologous region c is also located at the downstream end of double-stranded DNA fragment C and at the upstream end of double-stranded DNA fragment A. As a result, double-stranded DNA fragment A and double-stranded DNA fragment B are linked in homologous region a, double-stranded DNA fragment B and DNA C binds in homology region b, and double-stranded DNA fragment C and double-stranded DNA fragment A bind in homology region c. This makes it possible to obtain a cDNA in which double-stranded DNA fragment A, double-stranded DNA fragment B, and double-stranded DNA fragment C are linked in this order.

[0024] The homologous region and the complementary region can be any sequence, as long as individual strands having such a region can specifically hybridize with each other in the coupling reaction solution. Base pair length (bp), GC ratio, etc. in this area can be appropriately determined using the general method of designing probes and primers. Generally speaking, the base pair length of the homologous region should be sufficient to suppress non-specific hybridization and to properly bind the linear double-stranded DNA fragments of interest. On the other hand, if the base pairs of the homologous region are too long, then this may reduce the binding efficiency. In the present invention, the base pair length of the homologous region or the complementary region is preferably 10 base pairs (bp) or more, more preferably 15 bp. or more, and even more preferably 20 p. or more. The base pair length of the homologous region or complementary region is preferably 500 bp. or less, more preferably 300 p. or less, and even more preferably 200 p. or less.

[0025] In the method for producing DNA according to the invention, the length of DNA fragments associated with each other is not particularly limited. For example, the length of a linear double-stranded DNA fragment is preferably 50 bp. or more, more preferably 100 p. or more, and even more preferably, 200 p. or more. The length of a single stranded DNA fragment is preferably 50 bases or more, more preferably 100 bases or more, and even more preferably 200 bases or more. Double-stranded 325 kb DNA fragments can also be linked in the method for producing DNA according to the invention. The length of linked DNA fragments can vary depending on their types.

[0026] In the method for producing DNA according to the invention, all or part of the homologous region of linear double-stranded DNA fragments associated with each other must have a double-stranded structure in which two single-stranded DNAs hybridize with each other. That is, the linear double-stranded DNA fragment may be a full-length linear double-stranded DNA fragment without gaps or nicks, or it may be a linear double-stranded DNA fragment having a single-stranded structure at one or more positions. For example, a linked linear double-stranded DNA fragment may have a blunt end or a protruding end. In the method for producing DNA according to the invention, a linear double-stranded DNA fragment with a blunt end and a linear double-stranded DNA fragment with an overhanging end can be linked.

[0027] The molar ratio of each DNA fragment included in the reaction solution is preferably the same as the ratio of the number of molecules of each DNA fragment constituting the associated molecule of interest. By pairing a certain number of DNA fragments in the reaction system at the beginning of the coupling reaction, such a coupling reaction can be carried out more efficiently. Thus, for example, when linking DNA fragments of all different types, the molar concentrations of each DNA fragment contained in the reaction solution are preferably the same.

[0028] The total number of DNA fragments included in the reaction solution is not particularly limited. Since a sufficient amount of bound product can be easily obtained, the total concentration of DNA fragments contained in the reaction solution at the beginning of the coupling reaction is preferably 0.01 nM or more, more preferably 0.1 nM or more, and even more preferably, 0.3 nM or more. Since the binding efficiency is higher and suitable for binding a plurality of fragments, the total concentration of DNA fragments contained in the reaction solution at the beginning of the binding reaction is preferably 100 nM or less, more preferably 50 nM or less, even more preferably 25 nM or less, and particularly preferably 20 nM or less.

[0029] In the method for producing DNA according to the invention, the size of the bound molecule obtained by the binding reaction is not particularly limited. For example, the resulting bonded molecule is preferably 1,000 bases or more, more preferably 5,000 bases or more, even more preferably 10,000 bases or more, and even more preferably 20,000 bases or more. The method for producing DNA according to the invention makes it possible to obtain a linked molecule having a length of 300,000 bases or more, preferably 500,000 bases or more, and even more preferably 2,000,000 bases or more.

[0030] The exonuclease used in the present invention is an enzyme that sequentially hydrolyzes linear DNA from the 3'end or 5'end. The exonuclease used in the present invention is not particularly limited in type or biological origin, as long as it has an enzymatic activity that allows sequential hydrolysis from the 3'end or 5'end of linear DNA. Examples of enzymes that carry out sequential hydrolysis from the 3'-end (3'→5'-exonuclease) is a 3'→5'-exonuclease specific for linear double-stranded DNA, such as the exonuclease family III-AP (non-purine/non-pyrimidine) endonuclease and a 3'→5' exonuclease specific for single stranded DNA, such as the DnaQ superfamily protein. Exemplary endonucleases of the family III-AP endonucleases are exonuclease III (derived from Escherichia coli), ExoA (homolog of Bacillus subtilis exonuclease III), Mth212 (homolog of archaebacteria exonuclease III), and AP endonuclease I (human homolog of exonuclease III). Examples of DnaQ superfamily proteins are exonuclease I (derived from E. coli), exonuclease T (Exo T) (also known as RNase T), exonuclease X, DNA polymerase III epsilon subunit, DNA polymerase I, DNA polymerase II, DNA T7 α-polymerase, T4 DNA polymerase, Klenow 5 DNA polymerase, Phi29 DNA polymerase, ribonuclease III (RNase D), and oligoribonuclease (ORN). Examples of enzymes that carry out sequential hydrolysis of linear DNA from the 5'-end (5'→3'-exonuclease) are λ-exonuclease, exonuclease VIII, T5 exonuclease, T7 exonuclease and RecJ exonuclease.

[0031] The preferred exonuclease used in the present invention is the 3'→5'-exonuclease in terms of an effective balance between the ability to cleave linear double-stranded DNA fragments and the binding efficiency in the presence of RecA family recombinase proteins. Among these exonucleases, 3'→5'-exonuclease specific for linear double-stranded DNA is more preferred, an exonuclease of the AP exonuclease family III is even more preferred, and exonuclease III is particularly preferred.

[0032] In the present invention, the reaction solution preferably contains a linear double-stranded DNA-specific 3'→5'-exonuclease and a single-stranded DNA-specific 3'→5'-exonuclease as exonucleases. Binding efficiency is greatly improved by combining a 3'→5' exonuclease specific for single stranded DNA and a 3'→5' exonuclease specific for linear double stranded DNA compared to the efficiency when using only a 3'→5' exonuclease, specific for linear double-stranded DNA. It is assumed that the 3'-overhang formed in the bound molecule by the action of 3'→5'-exonuclease specific to linear double-stranded DNA and RecA is hydrolyzed by the action of 3'→5'-exonuclease specific to single-stranded DNA, which leads to improve binding efficiency. Since the binding efficiency can be greatly improved, the exonuclease contained in the reaction solution of the invention is preferably a combination of a family III-AP endonuclease exonuclease and a 3'→5' exonuclease specific for single-stranded DNA of one or more types. More preferred is a combination of a family III-AP endonuclease exonuclease and one or more types of DnaQ superfamily proteins. Particularly preferred is a combination of exonuclease III and exonuclease I or a combination of exonuclease III, exonuclease I and exonuclease T.

[0033] In the present invention, the concentration of the exonuclease in the coupling reaction reaction solution at the beginning of the coupling reaction is preferably, for example, 1-1000 mU/µl, more preferably 5-1000 mU/µl, more preferably 5-500 mU/µl, and even more preferably 10-150 mU/µl. In particular, if the exonuclease is a 3'→5'-exonuclease specific for linear double-stranded DNA, then the concentration of 3'→5'-exonuclease specific for linear double-stranded DNA in the reaction solution at the beginning of the coupling reaction is preferably, for example, 5 -500 mU/µl, more preferably 5-250 mU/µl, more preferably 5-150 mU/µl, and even more preferably 10-150 mU/µl. If the exonuclease is a 3'→5'-exonuclease specific for single-stranded DNA, then the concentration of 3'→5'-exonuclease specific for single-stranded DNA in the reaction solution at the beginning of the binding reaction is preferably, for example, 1-10000 mU/µl , more preferably 100-5000 mU/µl, and even more preferably 200-2000 mU/µl. If a linear double-stranded DNA-specific 3'→5'-exonuclease and a single-stranded DNA-specific 3'→5'-exonuclease are used in combination, the concentration of each exonuclease in the reaction solution at the start of the coupling reaction may be the preferred concentration each of the above exonucleases.

[0034] As used herein and herein, a RecA family recombinase protein means a protein having recombinase activity of the RecA family. This activity includes the function of polymerizing on single-stranded or double-stranded DNA to form a strand; hydrolytic activity for nucleoside triphosphates such as ATP (adenosine triphosphate); and the function of searching for a homologous region and effecting homologous recombination. Examples of RecA family recombinase proteins are prokaryotic RecA homologue, bacteriophage RecA homologue, archaebacteria RecA homologue, eukaryotic RecA homologue, and the like. Examples of prokaryotic RecA homologues are E. coli RecA; RecA derived from highly thermophilic bacteria such as Thermus bacteria such as Thermus thermophiles and Thermus aquaticus, Thermococcus bacteria, Pyrococcus bacteria, and Thermotoga bacteria; RecA derived from radiation resistant bacteria such as Deinococcus radiodurans. Examples of bacteriophage RecA homologues are the T4 UvsX phage. Examples of archaebacteria RecA homologues are RadA. Examples of eukaryotic RecA homologues are Rad51 and its paralog, and Dcm1. The amino acid sequences of these RecA homologues can be taken from databases such as NCBI (http://www.ncbi.nlm.nih.gov/).

[0035] The RecA family recombinase protein used in the present invention may be a wild-type protein or a variant thereof. A variant is a wild-type protein in which one or more mutations have been introduced, namely deletions, additions or substitutions of 1-30 amino acids, and which retains the recombinase activity of the RecA family. Examples of variants are variants with amino acid substitutions that improve the function of searching for homologous regions in wild-type proteins; variants with various labels added to the N-terminus or C-terminus of wild-type proteins, and variants with increased heat resistance (WO 2016/013592). As a label, for example, labels commonly used for expression or purification of recombinant proteins, such as His tag, HA tag (hemagglutinin tag), Myc tag and Flag tag, can be used. The term "wild-type RecA family recombinase protein" means a protein having the same amino acid sequence as the amino acid sequence of the RecA family recombinase protein that has been conserved in microorganisms isolated from natural sources.

[0036] The RecA family recombinase protein used in the present invention is preferably a variant that retains the RecA family recombinase protein. Examples of variants are the F203W mutant in which the 203 amino acid residue of E. coli RecA phenylalanine is replaced by tryptophan, and mutants in which the phenylalanine corresponding to E. coli RecA 203 phenylalanine is replaced by tryptophan in various RecA homologues.

[0037] In the present invention, the amount of the RecA family recombinase protein in the reaction solution for the coupling reaction is not particularly limited. In the present invention, the concentration of the RecA family recombinase protein in the reaction solution at the beginning of the coupling reaction is preferably, for example, 0.01-100 μM, more preferably 0.1-100 μM, even more preferably 0.1-50 μM, even more preferably 0.5-10 µM, and particularly preferably 1.0-5.0 µM.

[0038] Nucleoside triphosphates or deoxynucleoside triphosphates are needed to communicate RecA family recombinase activity to RecA family recombinase proteins. For this reason, the reaction solution for the coupling reaction according to the invention contains at least one nucleoside triphosphate or deoxynucleotide triphosphate. Since nucleoside triphosphate is contained in the reaction solution for the coupling reaction of the invention, it is preferable to use one or more compounds selected from the group consisting of dATP (deoxyadenosine triphosphate), dGTP (deoxyguanosine triphosphate), dCTP (deoxycytidine triphosphate), and dTTP (deoxythymidine triphosphate). It is particularly preferred to use dATP. The total amount of nucleoside triphosphate and deoxynucleotide triphosphate contained in the reaction solution is not particularly limited as long as it is sufficient for the RecA family recombinase protein to have RecA family recombinase activity. In the present invention, the concentration of nucleoside triphosphate or the concentration of deoxynucleotide triphosphate in the coupling reaction reaction solution at the beginning of this coupling reaction is preferably, for example, 1 μM or more, more preferably 10 μM or more, and even more preferably, 30 μM or more. On the other hand, if the concentration of the nucleoside triphosphate in the reaction solution is too high, the binding efficiency of a plurality of fragments may be slightly reduced. Therefore, the concentration of nucleoside triphosphate or the concentration of deoxynucleotide triphosphate in the reaction solution at the start of the coupling reaction is preferably, for example, 1000 μM or less, more preferably 500 μM or less, and even more preferably 300 μM or less.

[0039] Magnesium ions (Mg ²⁺ ) are required to communicate RecA family recombinase activity to the RecA family recombinase protein and to communicate exonuclease activity to the exonuclease. Therefore, the reaction solution for the coupling reaction according to the invention contains a source of magnesium ions. The magnesium ion source is a substance that transfers magnesium ions to the reaction solution. Examples are magnesium salts such as magnesium acetate [Mg(OAc) ₂ ], magnesium chloride [MgCl ₂ ], and magnesium sulfate [MgSO ₄ ]. The preferred source of magnesium ions is magnesium acetate.

[0040] In the present invention, the concentration of the magnesium ion source in the coupling reaction solution is not particularly limited as long as the RecA family recombinase protein has RecA family recombinase activity and the exonuclease has exonuclease activity. The concentration of the magnesium ion source in the reaction solution at the start of the coupling reaction is preferably, for example, 0.5 mM or more, and more preferably 1 mM or more. On the other hand, if the concentration of magnesium ions in the reaction solution is too high, then the exonuclease activity also becomes too high, and this may reduce the binding efficiency of a plurality of fragments. For this reason, the concentration of the magnesium ion source in the reaction solution at the beginning of the coupling reaction is preferably, for example, 20 mM or less, more preferably 15 mM or less, even more preferably 12 mM or less, and most preferably 10 mM. or less.

[0041] The reaction solution for the coupling reaction according to the invention is prepared, for example, by adding DNA fragments, a RecA family recombinase protein, an exonuclease, at least one nucleoside triphosphate or deoxynucleotide triphosphate, and a magnesium ion source to a buffer. The buffer solution is not particularly limited, provided it is suitable for use at pH 7-9, and preferably pH 8. Examples of buffers are Tris-HCl, Tris-OAc, Hepes-KOH, phosphate buffer, MOPS-NaOH, and tricine-HCl. The preferred buffer is Tris-HCl or Tris-OAc. The concentration of the buffer solution is not particularly limited and can be appropriately selected by a person skilled in the art. In the case of Tris-HCl or Tris-OAc, for example, the concentration of the buffer solution may be selected from a concentration of 10 mM-100 mM, preferably 10 mM-50 mM, and more preferably 20 mM.

[0042] In the present invention, if UvsX is used as the RecA family recombinase protein, it is preferable that the reaction solution for the coupling reaction also contains UvsY of T4 phage. UvsY is a mediator of homologous recombination in T4 phage. In the T4 phage, the single-stranded DNA first binds to gp32 (single-stranded DNA binding protein) to form the single-stranded DNA-gp32 complex. The single-stranded DNA then binds to uvsX, causing gp32 in the complex to be replaced by uvsX, resulting in homologous recombination. UvsY stimulates ssDNA and uvsX binding by destabilizing the ssDNA-gp32 interaction and stabilizing the ssDNA-uvsX interaction, resulting in an acceleration of the homologous recombination reaction (Bleuit et al., Proceedings of the National Academy of Sciences of the United States of America, 2001 , vol.98 (15), p.8298-8305). In addition, in the present invention, binding efficiency is also promoted by using UvsY together with UvsX.

[0043] The reaction solution for the binding reaction according to the invention contains a DNA fragment, a RecA family recombinant enzyme protein, an exonuclease, at least a nucleoside triphosphate or a deoxynucleotide triphosphate, and a source of magnesium ions. The reaction solution also preferably contains a nucleoside triphosphate or deoxynucleotide triphosphate regenerating enzyme and a substrate thereof. By regenerating nucleoside triphosphates or deoxynucleotide triphosphates in the reaction solution, a large number of DNA fragments can bind more efficiently. Examples of combinations of regenerating enzymes and their substrates for nucleoside triphosphate or deoxynucleotide triphosphate regeneration are the combination of creatine kinase and creatine phosphate, the combination of pyruvate kinase and phosphoenol pyruvate, the combination of acetate kinase and acetyl phosphate, the combination of polyphosphate kinase and polyphosphate, and the combination of nucleoside diphosphate kinase. and nucleoside triphosphate. The nucleoside triphosphate used as a substrate (source of phosphate replenishment) for the nucleoside diphosphate kinase may be any of ATP, GTP, CTP and UTP. In addition, an example of a regenerating enzyme is myokinase.

[0044] In the present invention, the concentrations of the nucleoside triphosphate regenerating enzyme and its substrate in the reaction solution for the coupling reaction are not particularly limited, as long as these concentrations are sufficient to regenerate the nucleoside triphosphate during the coupling reaction in the reaction solution. For example, when using creatine kinase and creatine phosphate, the concentration of creatine kinase in the reaction solution for the coupling reaction according to the invention is preferably 1-1000 ng/µl, more preferably 5-1000 ng/µl, even more preferably 5-500 ng/µl , and even more preferably 5-250 ng/µl. The concentration of creatine phosphate in the solution is preferably 0.4-20 mM, more preferably 0.4-10 mM, and even more preferably 1-7 mM.

[0045] If multiple fragments are linked in the desired order, then the base sequences of the homologous region or complementary region are preferably different for each combination of linked DNA fragments. However, under the same temperature conditions, a single-stranded homologous region having a high content of G (guanine base) and C (cytosine base) tends to form a secondary structure. On the other hand, a homologous region having a high content of A (adenine base) and T (thymine base) has low hybridization efficiency. Thus, the binding efficiency may decrease. By inhibiting the formation of the secondary structure of single-stranded DNA and stimulating specific hybridization, stimulation of binding of DNA fragments can be increased.

[0046] Therefore, it is preferable that a substance be added that inhibits the formation of a secondary structure of single-stranded DNA and promotes specific hybridization in the reaction solution for the coupling reaction of the invention. Examples of such substances are dimethyl sulfoxide (DMSO) and tetramethylammonium chloride (TMAC). DMSO inhibits the formation of secondary structure of GC-rich base pairs. TMAS stimulates specific hybridization. In the present invention, when a substance that promotes the formation of a secondary structure of single-stranded DNA and promotes specific hybridization is added to the binding reaction reaction solution, the concentration of such a substance is not particularly limited as long as the effect of promoting DNA fragment binding is achieved using such a substance. For example, when using DMSO as such a substance, the concentration of DMSO in the reaction solution for the coupling reaction according to the invention is preferably 5-30% by volume, more preferably 8-25% by volume, and even more preferably 8- 20% by volume. When using TMAC as such a substance, the concentration of TMAC in the reaction solution for the coupling reaction according to the invention is preferably 60-300 mM, more preferably 100-250 mM, and even more preferably 100-200 mM.

[0047] In the present invention, to the reaction solution for the coupling reaction, it is preferable that a substance imparting a macromolecular polymerization effect be added. The effect of macromolecular polymerization can stimulate the interaction of DNA molecules with each other and the binding of DNA fragments. Examples of such substances are polyethylene glycol (PEG) 200-20000, polyvinyl alcohol (PVA) 200-20000, dextran 40-70, Ficoll 70 and bovine serum albumin (BSA). In the present invention, when a substance imparting a macromolecular polymerization effect is added to the binding reaction reaction solution, the concentration of such a substance is not particularly limited as long as the substance promotes the binding of DNA fragments. For example, when using PEG8000 as such a substance, the concentration of this substance in the coupling reaction solution is preferably 2-20% by mass, more preferably 2-10% by mass, and even more preferably 4-6 % by weight.

[0048] In the present invention, the reaction solution for the coupling reaction may also contain an alkali metal ion source. An alkali metal ion source is a substance that introduces alkali metal ions into the reaction solution. In the present invention, the alkali metal ion contained in the reaction solution for the coupling reaction is preferably sodium ion (Na ⁺ ) or potassium ion (K ⁺ ). Examples of the alkali metal ion source are potassium glutamate [KGlu], potassium aspartate, potassium chloride, potassium acetate [KOAc], monosodium glutamate, sodium aspartate, sodium chloride, and sodium acetate. In the present invention, the alkali metal ion source contained in the coupling reaction reaction solution is preferably potassium glutamate or potassium acetate. Potassium glutamate is particularly preferred because it improves the binding efficiency of multiple moieties. The concentration of the alkali metal ion source in the reaction solution at the start of the coupling reaction is not particularly limited. For example, the concentration of the alkali metal ion source is preferably a concentration that can be adjusted to an alkali metal ion concentration in the reaction solution of 10 mM or more, preferably in the range of 30-300 mM, and even more preferably, within 50-150 mM.

[0049] In the present invention, the reaction solution for the coupling reaction may also contain a reducing agent. Examples of the reducing agent are dithiothreitol (DTT), β-mercaptoethanol (2-mercaptoethanol), tris(2-carboxyethyl)phosphine (TCEP) and glutathione. The preferred reducing agent is DTT. The reducing agent may be contained in the reaction solution at a concentration of 1.0-15.0 mM, and preferably 2.0-10.0 mM.

[0050] In the method for producing DNA according to the invention, the binding reaction is carried out by incubating the reaction solution for a predetermined period of time under isothermal conditions at a temperature at which the RecA family recombinant enzyme protein and the exonuclease in the reaction solution can have enzymatic activity. The reaction solution is prepared in a buffer solution containing DNA fragments of two or more species, a RecA family recombinant enzyme protein, a nucleoside triphosphate and a source of magnesium ions, and in addition, if necessary, an exonuclease, a set of enzymes for the regeneration of nucleoside triphosphate and their substrates; a substance that inhibits the formation of the secondary structure of single-stranded DNA and stimulates specific hybridization; a substance that communicates the effect of macromolecular polymerization; an alkali metal ion source; and a reducing agent. The coupling reaction temperature is preferably in the range of 25-48°C, and more preferably in the range of 27-45°C. In particular, if the length of the homologous region or the complementary region is 50 bases or more, then the coupling reaction temperature is preferably in the range of 30-45°C, more preferably in the range of 37-45°C, and even more preferably in the range 40-43°C. On the other hand, if the length of the homologous region or the complementary region is 50 bases or less, then the coupling reaction temperature is preferably in the range of 27-43°C, more preferably in the range of 27-37°C, and even more preferably in within 27-33°C. In the present description, the term "isothermal conditions" means conditions under which the temperature during the reaction is maintained within the range of ±3°C or ±1°C. The coupling reaction time is not particularly limited, and may be, for example, 15 minutes to 6 hours, and preferably 15 minutes to 2 hours.

[0051] As shown in FIG. 1, gaps and nicks are present in the bound molecule (linear or circular DNA) obtained by the coupling reaction. A gap means the absence of one or more adjacent nucleotides in double-stranded DNA. Nick means a gap resulting from the cleavage of a phosphodiester bond between adjacent nucleotides in double-stranded DNA. In the method for producing DNA according to the invention, after the coupling reaction, it is preferable to carry out gap and nick repair in the resulting coupled molecule using gap repair enzymes and dNTPs. After the gaps and nicks are repaired, the bound molecule can be converted into full-length double-stranded DNA.

[0052] In particular, the repair and nicking of the coupled molecule is carried out by adding the gap repair enzymes and dNTP to the reaction solution after the coupling reaction, followed by incubation for a predetermined period of time under isothermal conditions under which the group of gap repair enzymes will have enzymatic activity. . The types of gap repair enzymes are not particularly limited and may be of any biological origin, as long as they have the ability to repair gaps and nicks in double-stranded DNA. As gap repair enzymes, for example, a combination of an enzyme having DNA polymerase activity and an enzyme having DNA ligase activity can be used. When using DNA ligase derived from Escherichia coli as DNA ligase, the reaction solution contains NAD (nicotinamide adenine dinucleotide), which is its cofactor and is contained in a concentration in the range of 0.01-1.0 mM. Treatment with gap repair enzymes can be carried out, for example, at 25-40° for 5-120 minutes, and preferably for 10-60 minutes.

[0053] dNTP is a generic term for dATP, dGTP, dCTP, and dTTP. The concentration of dNTP contained in the reaction solution at the beginning of the repair reaction may be, for example, in the range of 0.01-1 mm, and preferably in the range of 0.05-1 mm.

[0054] It is also preferred that the associated molecule (linear or circular DNA) be further amplified to repair gaps and nicks. The method for amplifying a coupled molecule with gap and nick repair is not particularly limited. Amplification can be carried out by a general amplification method using linear or circular DNA as a template.

[0055] In the DNA production method of the invention, if the bound molecule obtained by performing the gap and nick repair reaction after the coupling reaction is linear, the bound molecule is preferably amplified by polymerase chain reaction (PCR). PCR can be carried out by a standard method.

[0056] In the DNA production method of the invention, if the bound molecule obtained by performing the gap and nick repair reaction after the coupling reaction is circular, the bound molecule is preferably amplified by rolling ring amplification (RCA). RCA can be done in a standard way.

[0057] In the method for producing DNA according to the invention, if the bound molecule obtained by performing the gap and nick repair reaction after the coupling reaction is circular and has an origin of replication sequence (chromosome origin (Oric)) capable of binding to an enzyme having DnaA activity, then the bound molecule is preferably amplified by the amplification method by replication cycle reaction (RCR). By carrying out RCR amplification using the bound molecule obtained immediately after the coupling reaction as a template, i.e., without performing the gap and nick repair reaction, it is possible to obtain a circular bound molecule consisting of full-length double-stranded DNA without gaps and nick as the amplification product.

[0058] For example, the known sequence of the origin of replication found in bacteria such as Escherichia coli and Bacillus subtilis can be taken from a public database such as NCBI. The sequence of the origin of replication can also be obtained by cloning a DNA fragment that can bind to an enzyme having DnaA activity and analyzing its base sequence.

[0059] In particular, the RCR amplification method can be carried out by preparing a reaction mixture and incubating the resulting reaction mixture. The reaction mixture includes a ring bound molecule obtained by a coupling reaction as a matrix; the first group of enzymes that catalyze the replication of circular DNA; the second group of enzymes that catalyze the binding reaction of Okazaki fragments and synthesize two sister circular DNA, consisting of catenan; a third group of enzymes that catalyze the separation of two sister cDNA, and dNTP. The two sister cDNAs of catenane are two cDNAs synthesized by DNA replication and in linked form.

[0060] An example of a first group of enzymes that catalyze the replication of circular DNA is the group of enzymes presented by Kaguni J.M. and Kornberg A. Cell 1984, 38: 183-90. In particular, examples of the first group of enzymes are one or more enzymes or groups of enzymes selected from the group consisting of an enzyme having DnaA; activity, nucleoid proteins of one or more types; an enzyme or group of enzymes having DNA gyrase activity; single-stranded DNA binding protein (SSB); an enzyme having helicase activity of the DnaB type; an enzyme having DNA helicase loading activity; an enzyme having DNA-primase activity; an enzyme having DNA-capturing activity; an enzyme or group of enzymes having DNA polymerase III* activity, and combinations of all of the aforementioned enzymes or groups of enzymes.

[0061] The biological origin of an enzyme having DnaA activity is not particularly limited, provided that such an enzyme has an initiation activity similar to that of DnaA, which is an Escherichia coli initiation protein, with DnaA derived from Escherichia coli. DnaA derived from Escherichia coli may be present as a monomer in the reaction solution at a concentration of, but not limited to, 1 nM - 10 mM, and preferably at a concentration of 1 nM - 5 mM, 1 nM - 3 mM, 1 nM - 1.5 mM, 1 nM - 1.0 mM, 1 nM - 500 nM, 50 nM - 200 nM or 50 nm - 150 nM.

[0062] A nucleotide protein is a protein within a nucleotide. The biological origin of the nucleotide protein of one or more types used in the present invention is not particularly limited, as long as it has an activity similar to that of the Escherichia coli nucleotide protein. Thus, for example, IHF derived from Escherichia coli, namely the complex of IhfA and/or IhfB (heterodimer or homodimer), or HU derived from Escherichia coli, namely the complex of HupA and HupB is preferably used. IHF derived from Escherichia coli may be contained as a hetero/homodimer in the reaction solution at a concentration in the range of 5 nM-400 nM. Preferably, IHF derived from Escherichia coli may be contained in the reaction solution at a concentration in the range of 5 nM-200 nM, 5 nM-100 nM, 5 nM-50 nM, 10 nM-50 nM, 10 nM-40 nM or 10 nM -30 nM, although these concentrations are not limited to the above limits. HU derived from Escherichia coli may be contained in the reaction solution at a concentration in the range of 1 nM - 50 nM, and preferably, it may be contained in this solution in a concentration in the range of 5 nM - 50 nM, or 5 nM - 25 nM, although these concentrations are not limited to the above limits.

[0063] The biological origin of an enzyme or group of enzymes having DNA gyrase activity is not particularly limited, provided that these enzymes have an activity similar to that of Escherichia coli DNA gyrase. Thus, for example, a complex of GyrA and GyrB derived from Escherichia coli is preferably used. Such a complex of GyrA and GyrB derived from Escherichia coli may be contained as a heterotetramer in the reaction solution at a concentration in the range of 20 nM - 500 nM, and preferably, it may be contained in the reaction solution at a concentration in the range of 20 nM - 400 nM, 20 nM - 300 nM, 20 nM - 200 nM, 50 nM - 200 nM or 100 nM - 200 nM, however, these concentrations are not limited to the above limits.

[0064] The biological origin of the single chain binding molecule (SSB) protein is not particularly limited, as long as it has an activity similar to that of the Escherichia coli single chain binding protein. Thus, for example, SSB derived from Escherichia coli is preferably used. Such an Escherichia coli derived SSB may be present as a homotetramer in the reaction solution at a concentration in the range of 20 nM - 1000 nM, and preferably it may be present in this solution in a concentration in the range of 20 nM - 500 nM, 20 nM - 300 nM . however, these concentrations are not limited to the above limits.

[0065] The biological origin of an enzyme having helicase activity of the DnaB type is not particularly limited, provided that such an enzyme has an activity similar to that of Escherichia coli DnaB. Thus, for example, DnaB derived from Escherichia coli is preferably used. Such a DnaB derived from Escherichia coli may be present as a homohexamer in the reaction solution at a concentration in the range of 5 nM - 200 nM, and preferably, it may be present in this solution in a concentration in the range of 5 nM - 100 mM, 5 nM - 50 mM or 5 nM - 30 nM, however, these concentrations are not limited to the above limits.

[0066] The biological origin of an enzyme having a DNA helicase loading activity is not particularly limited as long as such an enzyme has an activity similar to that of Escherichia coli DnaC. Thus, for example, DnaC derived from Escherichia coli is preferably used. Such a DnaC derived from Escherichia coli may be present as a homohexamer in the reaction solution at a concentration in the range of 5 nM - 200 nM, and preferably, it may be present in this solution in a concentration in the range of 5 nM - 100 nM, 5 nM - 50 nM or 5 nM - 30 nM, however, these concentrations are not limited to the above limits.

[0067] The biological origin of an enzyme having DNA primase activity is not particularly limited, provided that such an enzyme has an activity similar to Escherichia coli DnaG. Thus, for example, DnaG derived from Escherichia coli is preferably used. Such DnaG derived from Escherichia coli may be present as a monomer in the reaction solution at a concentration in the range of 20 nM - 1000 nM, and preferably, it may be present in this solution in a concentration in the range of 20 nM - 800 nM, 50 nM - 800 nM , 100nM-800nM, 200nM-800nM, 250nM-800nM, 250nM-500nM or 300nM-500nM, however, these concentrations are not limited to the above limits.

[0068] The biological origin of an enzyme having DNA capturing activity is not particularly limited, provided that such an enzyme has an activity similar to Escherichia coli DnaN. Thus, for example, DnaN derived from Escherichia coli is preferably used. Such a DnaN derived from Escherichia coli may be present as a homodimer in the reaction solution at a concentration in the range of 10 nM - 1000 nM, and preferably, it may be present in this solution in a concentration in the range of 10 nM - 800 nM, 10 nM - 500 nM , 20 nM - 500 nM, 20 nM - 200 nM, 30 nM - 200 nM or 30 nM - 100 nM, however, these concentrations are not limited to the above limits.

[0069] The biological origin of an enzyme or group of enzymes having the activity of DNA polymerase III* is not particularly limited, provided that such an enzyme or group of enzymes will have an activity similar to that of Escherichia coli DNA polymerase III* complex. Thus, for example, a group of enzymes is preferably used, including any of the enzymes DnaX, HolA, HolB, HoLC, HoLD, DnaE, DnaQ and HolE, derived from Escherichia coli, preferably, a group of enzymes, including a complex of enzymes DnaX, HolA, HolB and DnaE, derived from Escherichia coli, and more preferably, a group of enzymes, including a complex of enzymes DnaX, HolA, HolB, HoLC, HoLD, DnaE, DnaQ and HolE derived from Escherichia coli. Such a DNA polymerase III* complex derived from Escherichia coli may be present as a heteromultimer in the reaction solution at a concentration in the range of 2 nM - 50 nM, and preferably, it may be present in this solution in a concentration in the range of 2 nM - 40 nM, 2 nM - 30 nM, 2 nM - 20 nM, 5 nM - 40 nM, 5 nM - 30 nM or 5 nM - 20 nM, however, these concentrations are not limited to the above limits.

[0070] Examples of the second groups of enzymes that catalyze the maturation of the Okazaki fragment and synthesize two sister circular DNAs consisting of catenane can be, for example, one or more enzymes selected from the group consisting of an enzyme having DNA polymerase I activity; an enzyme having DNA ligase activity; and an enzyme having RNase H activity, or a combination of these enzymes.

[0071] The biological origin of an enzyme having the activity of DNA polymerase I is not particularly limited, provided that such an enzyme has an activity similar to that of Escherichia coli DNA polymerase I. Thus, for example, DNA polymerase I derived from Escherichia coli is preferably used. Such DNA polymerase I derived from Escherichia coli may be present as a monomer in the reaction solution at a concentration in the range of 10 nM - 200 nM, and preferably, it may be present in this solution at a concentration in the range of 20 nM - 200 nM, 20 nM - 150 nM, 20 nM - 100 nM, 40 nM - 150 nM, 40 nM - 100 nM or 40 nM - 80 nM, however, these concentrations are not limited to the above limits.

[0072] The biological origin of an enzyme having DNA ligase activity is not particularly limited as long as such an enzyme has an activity similar to that of Escherichia coli DNA ligase. Thus, for example, DNA ligase derived from Escherichia coli or T4 phage DNA ligase is preferably used. Such a DNA ligase derived from Escherichia coli may be present as a monomer in the reaction solution at a concentration in the range of 10 nM - 200 nM, and preferably, it may be present in this solution in a concentration in the range of 15 nM - 200 nM, 20 nM - 200 nM, 20 nM - 150 nM, 20 nM - 100 nM or 20 nM - 80 nM, however, these concentrations are not limited to the above limits.

[0073] The biological origin of an enzyme having RNase H activity is not particularly limited as long as such an enzyme has an RNA-DNA hybrid RNA strand degrading activity. Thus, for example, an RNase H derived from Escherichia coli is preferably used. Such RNase H derived from Escherichia coli may be present as a monomer in the reaction solution at a concentration in the range of 0.2 nM - 200 nM, and preferably, it may be present in this solution in a concentration in the range of 0.2 nM - 200 nM, 0.2 nM - 100 nM, 0.2 nM - 50 nM, 1 nM - 200 nM, 1 nM - 100 nM, 1 nM - 50 nM or 10 nM - 50 nM, however, these concentrations are not limited to the above limits.

[0074] An example of a third group of enzymes that catalyze the separation of two sister cDNAs is the group of enzymes represented, for example, as the group of enzymes described by Peng H & Marians KJ PNAS 1993, 90: 8571-8575. In particular, examples of the third group of enzymes are one or more enzymes selected from the group consisting of an enzyme having topoisomerase IV activity; an enzyme having topoisomerase III activity; and an enzyme having helicase activity of the RecQ type; or combinations of the aforementioned enzymes.

[0075] The biological origin of an enzyme having the activity of topoisomerase III is not particularly limited, provided that such an enzyme has an activity similar to that of Escherichia coli topoisomerase III. Thus, for example, topoisomerase III derived from Escherichia coli is preferably used. Such topoisomerase III derived from Escherichia coli may be present as a monomer in the reaction solution at a concentration in the range of 20 nM - 500 nM, and preferably, it may be present in this solution in a concentration in the range of 20 nM - 400 nM, 20 nM - 300 nM, 20 nM - 200 nM, 20 nM - 100 nM, or 30 nM - 80 nM, however, these concentrations are not limited to the above limits.

[0076] The biological origin of an enzyme having helicase activity of the RecQ type is not particularly limited, provided that such an enzyme has an activity similar to that of Escherichia coli RecQ. Thus, for example, RecQ derived from Escherichia coli is preferably used. Such a RecQ derived from Escherichia coli may be present as a monomer in the reaction solution at a concentration in the range of 20 nM - 500 nM, and preferably, it may be present in this solution in a concentration in the range of 20 nM - 400 nM, 20 nM - 300 nM , 20 nM - 200 nM, 20 nM - 100 nM, or 30 nM - 80 nM, however, these concentrations are not limited to the above limits.

[0077] The biological origin of an enzyme having the activity of topoisomerase IV is not particularly limited, provided that such an enzyme has an activity similar to that of Escherichia coli topoisomerase IV. Thus, for example, topoisomerase IV is preferably used, which is derived from Escherichia coli and is a complex of ParC and ParE. Such a topoisomerase IV derived from Escherichia coli may be present as a heterotetramer in the reaction solution at a concentration in the range of 0.1 nM - 50 nM, and preferably, it may be present in this solution in a concentration in the range of 0.1 nM - 40 nM, 0.1 nM - 30 nM, 0.1 nM - 20 nM, 1 nM - 40 nM, 1 nM - 30 nM, 1 nM - 20 nM, 1 nM - 10 nM, or 1 nM - 5 nM, however, these concentrations are not limited to the above limits.

[0078] The first, second and third groups of enzymes above may be enzymes that are commercially available or may be extracted from microorganisms and purified if necessary. Extraction and purification of enzymes from microorganisms can be carried out, if necessary, by methods known to those skilled in the art.

[0079] If enzymes other than the above-described enzymes derived from Escherichia coli are used as the first, second, and third groups of enzymes, each of these enzymes can be used at a concentration within the corresponding values of the unit of enzyme activity, and such a concentration should be similar to the concentration of the above enzyme derived from Escherichia coli.

[0080] As the dNTP contained in the reaction mixture used in the RCR amplification method, dNTPs similar to those used in the DNA production method of the invention can be used.

[0081] If necessary, the reaction mixture obtained by the method of RCR amplification, may also contain a source of magnesium ions, a source of alkali metal ions and ATP.

[0082] In the RCR amplification method, the concentration of ATP contained in the reaction mixture at the beginning of the reaction may be, for example, in the range of 0.1-3 mM, preferably in the range of 0.1-2 mM, more preferably in the range 0.1-1.5 mm, and even more preferably, in the range of 5-1.5 mm.

[0083] As a source of magnesium ions included in the reaction mixture used in the RCR amplification method, the same substances used in the DNA production method of the invention can be used. In the RCR amplification method, the concentration of the magnesium ion source contained in the reaction mixture at the beginning of the reaction may be, for example, the concentration required to introduce 5-50 mM magnesium ions into the reaction mixture.

[0084] As a source of alkali metal ions included in the reaction mixture used in the RCR amplification method, the same substances used in the DNA production method of the invention can be used. In the RCR amplification method, the concentration of the alkali metal source contained in the reaction mixture at the beginning of the reaction may be, for example, the concentration necessary to introduce alkali metal ions into the reaction solution in the range of 100 mM or more, and preferably 100 mM-300 mm, however, these concentrations are not limited to the above limits.

[0085] In the RCR amplification method, the amount of bound molecule contained in the reaction mixture is not particularly limited. For example, at the beginning of the reaction, the bound molecule may be present in the mixture at a concentration of 10 ng/µl or less, 5 ng/µl or less, 1 ng/µl or less, 0.8 ng/µl or less, 0.5 ng /µl or less, or 0.3 ng/µl or less.

[0086] When the resulting reaction mixture is incubated under isothermal conditions at a predetermined temperature, only the circular DNA containing the replication origin sequence capable of binding to an enzyme having DnaA activity is amplified. The reaction temperature in RCR amplification is not particularly limited, as long as the DNA replication reaction takes place. More specifically, the temperature may be, for example, in the range of 20-80°C, 25-50°C or 25-40°C, and such a temperature is the optimum temperature for DNA polymerase. The reaction time in RCR amplification can be appropriately selected depending on the amount of the amplification product of the circular linked target molecule. The reaction time may be, for example, from 30 minutes to 24 hours.

[0087] RCR amplification can also be carried out by incubating the resulting reaction mixture according to a thermal cycle that is repeated with incubation at 30°C or higher and with incubation at 27°C or lower. Incubation at 30°C or higher is not particularly limited, provided that such a temperature is a temperature within a range sufficient to initiate replication of cDNA including oriC. For example, the temperature may be 30-80°C, 30-50°C, 30-40°C or 37°C. Incubation at 30°C or higher can be carried out for a period of time from 10 seconds to 10 minutes per cycle, however, this period of time is not particularly limited. Incubation at 27° C. or below is not particularly limited as long as it is carried out at a temperature at which replication initiation is suppressed and DNA elongation reaction occurs. For example, the temperature may be 10-27°C, 16-25°C or 24°C. Incubation at 27° C. or lower may be preferably carried out depending on the length of the cDNA to be amplified, but is not particularly limited. So, for example, incubation can be carried out for 1-10 seconds per 1000 bases per cycle. The number of thermal cycles is not particularly limited, but may be 10 to 50 cycles, 20 to 40 cycles, 25 to 35 cycles, or 30 cycles.

[0088] The bound molecule obtained by the coupling reaction is preferably thermally incubated at 50°C-70°C followed by rapid cooling before being used in the gap and nick repair reaction and for RCR amplification as a template. The heat treatment time is not particularly limited, and may be, for example, 1-15 minutes, and preferably 2-10 minutes. The quench temperature is not particularly limited, and is, for example, 10°C or less, and preferably 4°C or less. The cooling rate is preferably 50°C/minute or more, more preferably 70°C/minute or more, and even more preferably 85°C/minute or more. For example, the reaction mixture in the container can be quenched after heat treatment by placing it directly on ice or by contacting a metal block brought to 4° C. or less.

[0089] The reaction solution obtained immediately after the binding reaction contains bound molecules obtained by non-specific binding. Non-specific binding can be subjected to dissociation by heat treatment and rapid cooling of the reaction solution. For this reason, gap and nick repair reactions and RCR amplification reactions are carried out using the bound molecule as a template after heat treatment and rapid cooling. This leads to suppression of non-specific binding, whereby full-length double-stranded DNA of the associated molecule of interest can be efficiently obtained.

[0090] In the method for producing DNA according to the invention, amplification of a linear or circular linked molecule obtained by a binding reaction is carried out in a microorganism by introducing the linked molecule into it. This can be done using the enzyme of this microorganism or the like. The bonded molecule introduced into the microorganism may be the bonded molecule before the gap and nick repair reaction or the bonded molecule after the repair reaction. Even when a coupled molecule having a gap and nick is directly introduced into a microorganism, the bound molecule in full-length double-stranded DNA without gaps and nick can be obtained as an amplification product. Examples of microorganisms into which the linked molecule is introduced are Escherichia coli, Bacillus subtilis, actinomycetes, archaebacteria, yeasts, filamentous fungi, and the like. The introduction of a bound molecule into a microorganism can be carried out by a standard method such as the electroporation method. The amplified bound molecule can be isolated from the microorganism by a standard method.

[0091] DNA Fragment Binding Kit

The kit for linking DNA fragments according to the invention is a kit for linking two or more types of DNA fragments to each other in areas where the base sequences are homologous, to obtain linear or circular DNA, and contains a RecA family recombinase protein. The kit, if used to link linear double-stranded DNA fragments, also preferably contains an exonuclease. The RecA family recombinase protein and the exonuclease contained in the kit are added to a solution containing two or more types of linked DNA fragments. Thus, the method for producing DNA according to the invention can be carried out in a simpler manner, and in this case, the associated molecule of interest can be easily obtained. Since the kit contains a RecA family recombinase protein, enzymes similar to those used in the method for producing DNA according to the invention can be used. The exonuclease contained in the kit is preferably a 3'→5' exonuclease. More preferably, the kit contains a 3'→5' exonuclease specific for linear double stranded DNA, and even more preferably a 3'→5' exonuclease specific for linear double stranded DNA and a 3'→5' exonuclease specific for single stranded DNA. DNA.

[0092] The kit for binding DNA fragments according to the invention also preferably includes a regenerating enzyme for nucleoside triphosphate or deoxynucleotide triphosphate and its substrate. The DNA fragment binding kit of the invention may include one or more substances selected from the group consisting of nucleoside triphosphate, deoxynucleotide triphosphate, magnesium ion source, alkali metal ion source, dimethyl sulfoxide, tetramethylammonium chloride, polyethylene glycol, dithiothreitol, and buffer. Any of these substances used in the method for obtaining DNA according to the invention can be used in the form indicated above.

[0093] The kit for binding DNA fragments according to the invention also preferably includes a document that describes the protocol for carrying out the method of obtaining DNA according to the invention using this kit. This protocol may be printed on the surface of the container containing this kit.

Examples

[0094] The present invention is described in more detail below with reference to the Examples, which are not considered as limiting the scope of the invention.

[0095] [Example 1]

Linear double-stranded DNA fragments of two or more types were joined using a RecA family recombinase protein and a 3'→5' exonuclease. In this reaction, the influence of the concentration of magnesium ion source and ATP in the reaction solution was investigated.

[0096] DCW1-DCW7 (SEQ ID NO: 1 - SEQ ID NO: 7), which are 591 bp linear double-stranded DNA fragments, were used as linked linear double-stranded DNA fragments. The region from the end to 60 bases of each linear double-stranded DNA fragment is the homologous region. That is, the 60 base region from base 532 to base 591 in DCW1 is a homologous region for binding to DCW2 and consists of the same base sequence as the 60 base sequence from base 1 to base 60 in DCW2. The 60 base region from base 532 to base 591 in DCW2 is a homologous region for binding to DCW3 and consists of the same base sequence as the 60 base sequence from base 1 to base 60 in DCW3. The 60 base region from base 532 to base 591 in DCW3 is a homologous region for binding to DCW4 and consists of the same base sequence as the 60 base sequence from base 1 to base 60 in DCW4. The 60 base region from base 532 to base 591 in DCW4 is a homologous region for binding to DCW5 and consists of the same base sequence as the 60 base sequence from base 1 to base 60 in DCW5. The 60 base region from base 532 to base 591 in DCW5 is a homologous region for binding to DCW6 and consists of the same base sequence as the 60 base sequence from base 1 to base 60 in DCW6. The 60 base region from base 532 to base 591 in DCW6 is a homologous region for binding to DCW7 and consists of the same base sequence as the 60 base sequence from base 1 to base 60 in DCW7.

[0097] Wild-type E. coli RecA (SEQ ID NO: 61) was used as the RecA family recombinase protein, and exonuclease III was used as the 3'→5'-exonuclease.

[0098] Specifically, reaction solutions were first prepared consisting of 1 nM each of DCW1-DCW7, 1 μM RecA, 40 mU/μs exonuclease III, 20 mM Tris-HCl (pH 8.0), 4 mM DTT, 1 mM or 10 mM magnesium acetate, 30 µM, 100 µM, 300 µM or 1000 µM ATP, 4 mM creatine phosphate, 20 ng/µl creatine kinase, 50 mM potassium glutamate, 150 mM TMAS, 5% by weight PEG8000 and 10% by volume DMSO . Then, these reaction solutions were incubated at 42°C for 2 hours to carry out the coupling reaction. 1 µl of the reaction solution after completion of the reaction was subjected to agarose gel electrophoresis, and the separated bands were stained with SYBR green (registered trademark).

[0099] The staining results are shown in FIG. 2. On this figure "500 bp" denotes a lane where a 500 bp DNA ladder marker was used. - 5 kb (total 10) at intervals of 500 p. In this figure, "7 frag" denotes the band of the bound molecule in which all seven fragments of DCW1-DCW7 have been linked.

[0100] From the reaction solutions with 1 mM magnesium acetate, in the reaction solution with 30 μM ATP, the bound molecule was almost not detected, and in the reaction solutions with 100 μM, 300 μM and 1000 μM ATP, bands of bound molecules of 6 types were detected, which extended from bound molecule of 2 fragments to bound molecule of 7 fragments. When comparing the results obtained for reaction solutions with 100 μM, 300 μM, and 1000 μM ATP, it was found that the reaction solution with 100 μM ATP contained the largest amount of the bound molecule obtained by binding fragments of all 7 types, and bands of unbound fragments were not detected. In contrast, in the reaction solution with 1000 μM ATP, the band of the bound molecule of 7 fragments was very weak, and many fragments remained unbound.

On the other hand, in reaction solutions with 10 mM magnesium acetate, bands of 6 types of bound molecules from a bound molecule of 2 fragments to a bound molecule of 7 fragments were detected in reaction solutions with 30 μM and 100 μM ATP. In reaction solutions with 300 μM and 1000 μM ATP, bands of bound molecules from a bound molecule of 2 fragments to a bound molecule of 4 fragments were detected, but bands of bound molecules with bound 5 or more fragments were not detected, and many fragments remained unbound.

Of all the samples, the amount of the obtained bound molecule of 7 fragments was the largest in the reaction solution with 1 mm magnesium acetate and 100 mm ATP. These results allow us to draw the following conclusions: for the binding of many fragments, the balance between the concentration of magnesium ions and ATP in the reaction solution plays an important role. And if the concentration of ATP is too high, it can lead to inhibition of the binding reaction.

[0101] [Example 2]

Linear double-stranded DNA fragments of two or more types were joined using a RecA family recombinase protein and a 3'→5' exonuclease. In this reaction, the influence of the concentration of PEG 8000 and the ATP-regenerating system in the reaction solution was investigated.

[0102] Lter1 - Lter5 (SEQ ID NO: 54 - SEQ ID NO: 58), which are linear double-stranded DNA fragments of 3100 bp in size. or 2650 bp, were used as linked linear double-stranded DNA fragments. The region from the end to 60 bases of each linear double-stranded DNA fragment is the homologous region. That is, the 60 base region from base 3041 to base 3100 in Lter1 is a homologous region for binding to Lter2 and consists of the same base sequence as the 60 base sequence from base 1 to base 60 in Lter2. The 60 base region from base 3041 to base 3100 in Lter2 is a homologous region for binding to Lter3 and consists of the same base sequence as the 60 base sequence from base 1 to base 60 in Lter3. The 60 base region from base 3041 to base 3100 in Lter3 is a homologous region for binding to Lter4 and consists of the same base sequence as the 60 base sequence from base 1 to base 60 in Lter4. The 60 base region from base 3041 to base 3100 in Lter4 is a homologous region for binding to Lter5 and consists of the same base sequence as the 60 base sequence from base 1 to base 60 in Lter5.

[0103] E. coli F203W RecA mutant was used as the RecA family recombinase protein, and exonuclease III was used as the 3'→5' exonuclease. Creatine kinase was used as an ATP-regenerating enzyme, and creatine phosphate was used as a substrate.

[0104] In particular, reaction solutions were first prepared consisting of 0.03 nM each Lter1-Lter5, 1 μM F203W RecA mutant, 40 mU/μl exonuclease III, 20 mM Tris-HCl (pH 8.0), 10 mM acetate magnesium, 100 μM ATP, 4 mM creatine phosphate, 20 ng/μl creatine kinase, 150 mM potassium acetate, and 0%, 2%, 5% or 10% by weight PEG8000. In this case, the reaction solutions were prepared separately in a similar way, except that they did not contain creatine phosphate and creatine kinase. Then, these reaction solutions were incubated at 30° C. for 30 minutes to carry out the coupling reaction. 4 μl of the reaction solution after completion of the reaction was subjected to agarose gel electrophoresis, and the separated bands were stained with SYBR green.

[0105] The staining results are shown in FIG. 3. In this figure, "5 frag" means the band of the bound molecule in which all five fragments of Lter1-Lter5 have been linked. Among the reaction solutions containing no creatine phosphate and creatine kinase, bands of a bound molecule of 2 fragments and a bound molecule of 3 fragments were detected in a reaction solution with 0 wt % PEG8000, and bands of bound molecules with 4 or more linked fragments were not detected. In contrast, bands of a 4-fragment bound molecule and a 5-fragment bound molecule were also detected in a reaction solution with a high concentration of PEG8000. On the other hand, among reaction solutions containing creatine phosphate and creatine kinase, 2-fragment bound molecule, 3-fragment bound molecule, and 4-fragment bound molecule were detected in reaction solutions with 0 wt% PEG8000, and the 5-fragment bound molecule band was not detected. In contrast, a 5 fragment bound molecule band was also detected in the reaction solution with a high concentration of PEG8000. As a result of comparing reaction solutions with 0% by weight PEG8000, it was found that the reaction solution with an ATP-regenerating system is more likely to form a linked molecule from multiple fragments. In the reaction solution without creatine phosphate and creatine kinase and in the reaction solution containing these compounds, the amount of the produced bound molecule of 5 fragments in the reaction solution with PEG exceeded the amount of this molecule in the reaction solution without PEG. Based on these results, PEG was found to stimulate binding. Of all these samples, the reaction solution with creatine phosphate, with creatine kinase and 5% by weight PEG8000 had the least number of unbound fragments, and the highest number of bound molecule of 5 fragments.

[0106] [Example 3]

Linear double-stranded DNA fragments of two or more types were joined using a RecA family recombinase protein and a 3'→5' exonuclease. In this reaction, the influence of the concentration of DMSO in the reaction solution was investigated.

[0107] DCW1-DCW5 (SEQ ID NO: 1 - SEQ ID NO: 5) were used as linked linear double-stranded DNA fragments. The F203W RecA mutant of E. coli was used as the RecA family recombinase protein, and exonuclease III was used as the 3'→5'-exonuclease.

[0108] In particular, reaction solutions were first prepared consisting of 1 nM each of DCW1-DCW5, 1 μM F203W RecA mutant, 40 mU/μl exonuclease III, 20 mM Tris-HCl (pH 8.0), 4 mM DTT, 10 mM magnesium acetate, 100 µM ATP, 150 mM potassium acetate, 5% by weight PEG8000, 0%, 1%, 3%, or 10% by volume DMSO. Then, these reaction solutions were incubated at 30° C. for 30 minutes to carry out the coupling reaction. 2 μl of the reaction solution after completion of the reaction was subjected to agarose gel electrophoresis, and the separated bands were stained with SYBR green.

[0109] The staining results are shown in FIG. 4(a). In this figure, "MK3" means the lane where the DNA ladder marker was used, and "In" means the lane where 2 μl of a solution containing 1 nM of each of DCW1-DCW5 is used. As a result, a bound molecule band in which all five fragments were bound was detected in all samples after the coupling reaction. The reaction solution with 10% v/v DMSO had more bound molecule of 5 fragments than the other reaction solutions.

[0110] Then the reaction solutions were prepared in a similar manner, except that the concentration of DMSO was 0% by volume, 10% by volume, 20% by volume or 40% by volume, and these reaction solutions were incubated at 30°C for 30 minutes for the coupling reaction. 2 μl of the reaction solution after completion of the reaction was subjected to agarose gel electrophoresis, and the separated bands were stained with SYBR green.

[0111] The staining results are shown in FIG. 4(b). In this figure, "500 bp ladder" means the lane where the DNA ladder marker was used in Example 1, and "In" means the same lane as in FIG. 4(a). As a result, the level of production of the bound molecule, in which all five fragments were linked to each other, increased markedly in the reaction solution with 10% by volume or 20% by volume of DMSO, but this band was not detected, and binding was inhibited in the reaction solution with 40% by volume of DMSO. Based on these results, the following conclusions were drawn. The binding reaction was stimulated when the content of DMSO was 5% by volume or more. In contrast, if the DMSO concentration was too high then the binding reaction was inhibited.

[0112] [Example 4]

Linear double-stranded DNA fragments of two or more types were joined using a RecA family recombinase protein and a 3'→5' exonuclease. In this reaction, the influence of the concentration of TMAC in the reaction solution was investigated.

[0113] DCW1-DCW7 (SEQ ID NO: 1 - SEQ ID NO: 7) were used as linked linear double-stranded DNA fragments. The E. coli F203W RecA mutant was used as the RecA family recombinase protein, and exonuclease III was used as the 3'→5'-exonuclease.

[0114] Specifically, reaction solutions were first prepared consisting of 1 nM each of DCW1-DCW7, 1 μM F203W RecA mutant, 40 mU/μl exonuclease III, 20 mM Tris-HCl (pH 8.0), 4 mM DTT, 10 mM magnesium acetate, 100 µM ATP, 4 mM creatine phosphate, 20 ng/µl creatine kinase, 150 mM potassium glutamate, 5% w/w PEG8000, 10% v/v DMSO and 0 mM, 15 mM, 30 mM, 60 mM or 100 mM TMAS. Then, these reaction solutions were incubated at 37°C for 2 hours to carry out the coupling reaction. 1.5 μl of the reaction solution after completion of the reaction was subjected to agarose gel electrophoresis, and the separated bands were stained with SYBR green.

[0115] The staining results are shown in FIG. 5(a). In this figure, "500 bp ladder" means the lane where the DNA ladder marker in Example 1 was used, and "In" means the lane where 1 μl of a solution containing 1 nM of each of DCW1-DCW7 is used. As a result, a bound molecule band in which all seven fragments were bound was detected in all samples after the coupling reaction. In particular, the amount of the bound molecule of 7 fragments in the reaction solution with 100 mm TMAS was markedly higher than the amount observed in other reaction solutions.

[0116] Then, the reaction solutions were prepared in a similar manner, except that the concentration of TMAC was 60 mM, 100 mM, 150 mM, 200 mM, or 250 mM, and the concentration of potassium glutamate was 50 mM. These reaction solutions were incubated at 42°C for 2 hours to carry out the coupling reaction. 1.9 μl of the reaction solution after completion of the reaction was subjected to agarose gel electrophoresis, and the separated bands were stained with SYBR green.

[0117] The staining results are shown in FIG. 5(b). As a result, the reaction solutions with 100 mM - 200 mM TMAS had a clearly higher content of the bound molecule of 7 fragments than the reaction solution with 60 mM TMAS, and their binding efficiency increased. The reaction solution with 150 mM TMAS had the most bound molecule of 7 fragments. On the other hand, in the reaction solution with 250 mM TMAC, the number of bound molecule of 7 fragments was less than in the reaction solution with 60 mM TMAS, and many unbound fragments remained. Based on this result, it was concluded that binding was stimulated by TMAS in an amount of 100-200 mM.

[0118] [Example 5]

Linear double-stranded DNA fragments of two or more types were joined using a RecA family recombinase protein and a 3'→5' exonuclease. In this reaction, the influence of alkali metal ion sources in the reaction solution was investigated.

[0119] DCW1-DCW7 or DCW1-DCW5 were used as linked linear double-stranded DNA fragments. The F203W RecA mutant of E. coli was used as the RecA family recombinase protein, and exonuclease III was used as the 3'→5'-exonuclease.

[0120] Specifically, reaction solutions were first prepared consisting of 1 nM each of DCW1-DCW5, 1 μM RecA mutant F203W, 40 mU/μl exonuclease III, 20 mM Tris-HCl (pH 8.0), 4 mM DTT, 10 mM magnesium acetate, 100 µM ATP, 4 mM creatine phosphate, 20 ng/µl creatine kinase, 0 mM, 50 mM, 75 mM, 100 mM, 125 mM or 150 mM potassium acetate, 5% by weight PEG8000, and 10% by weight volume of DMSO. These reaction solutions were prepared in a similar manner, except that they contained 150 mM potassium glutamate instead of potassium acetate. Then the reaction solutions were incubated at 30°C for 2 hours to carry out the binding reaction. 2 μl of the reaction solution after completion of the reaction was subjected to agarose gel electrophoresis, and the separated bands were stained with SYBR green.

[0121] The staining results are shown in FIG. 6. The bound molecule band in which all five fragments were bound was not detected in reaction solutions containing no alkali metal ion sources, and was detected in all reaction solutions containing potassium acetate or potassium glutamate. In particular, the band of unbound fragments in the reaction solution with potassium glutamate was weaker than in the reaction solution with potassium acetate. Thus, it has been suggested that potassium glutamate can significantly increase binding efficiency compared to potassium acetate.

[0122] Then, the reaction solutions were prepared in a similar manner, except that DCW1-DCW7 were used as linked linear double-stranded DNA fragments, and the concentration of each was 1 nM, potassium glutamate was used as the source of alkali metal ions, and the concentration potassium glutamate was 50 mM or 150 mM. These reaction solutions were incubated at 37°C, 42°C or 45°C for 1 hour to carry out the coupling reaction. 1 μl of the reaction solution after completion of the reaction was subjected to agarose gel electrophoresis, and the separated bands were stained with SYBR green.

[0123] The staining results are shown in FIG. 7. In this figure, "500 bp ladder" means the lane where the DNA ladder marker in Example 1 was used, and "In" means the lane where 1 μl of a solution containing 1 nM of each of DCW1-DCW7 is used. . As a result, at reaction temperatures of 37°C and 42°C, the reaction solutions with 50 mM potassium glutamate showed greater binding efficiency than the reaction solution with 150 mM potassium glutamate, and a band of a bound molecule was detected in which all seven fragments were bound. Based on these results, it was found that the binding reaction can be inhibited if the concentration of potassium glutamate is too high. In the reaction solutions incubated at 45°C, the band of the bound molecule of 7 fragments was not detected even if the concentration of potassium glutamate was 50 mM. The number of bound molecule of 7 fragments was the largest in the reaction solution, which contained 50 mm potassium glutamate and was incubated at 42°C.

[0124] [Example 6]

Linear double stranded DNA fragments of two or more types were linked using a RecA family recombinase protein and a 3'→5' exonuclease. In this reaction, the influence of the molar ratio of each linear double-stranded DNA fragment in the reaction solution was investigated.

[0125] DCW1-DCW7 were used as linked linear double-stranded DNA fragments. The E. coli F203W RecA mutant was used as the RecA family recombinase protein, and exonuclease III was used as the 3'→5'-exonuclease.

[0126] In particular, reaction solutions were first prepared consisting of 1 nM each of DCW1-DCW7, 1 μM F203W RecA mutant, 40 mU/μl exonuclease III, 20 mM Tris-HCl (pH 8.0), 4 mM DTT, 10 mM magnesium acetate, 100 μM ATP, 4 mM creatine phosphate, 20 ng/μl creatine kinase, 50 mM potassium acetate, 5% by weight PEG8000, and 10% by volume DMSO. These reaction solutions were prepared in a similar manner, except that the DCW3 concentration was 2 nM. Then the reaction solutions were incubated at 42°C for 2 hours to carry out the coupling reaction. 1 μl of the reaction solution after completion of the reaction was subjected to agarose gel electrophoresis, and the separated bands were stained with SYBR green.

[0127] The staining results are shown in FIG. 8. In this figure, "500 bp ladder" means the lane where the DNA ladder marker in Example 1 was used, and "In" means the lane where 1 μl of a solution containing 1 nM of each of DCW1-DCW7 is used. . The term "equal" means a lane where a reaction solution containing 1 nM of each of DCW1 ~ DCW7 is used. "2-fold excess of the 3rd fragment" means a lane where there is a reaction solution containing 1 nM of each of DCW1-DCW7, except for DCW3, the concentration of which was 2 nM DCW3. As a result, a bound molecule of 7 fragments was obtained in the entire reaction solution. However, in the reaction solution containing only twice the concentration (two moles) of DCW3, the amount of bound 7-piece molecule decreased, and the number of bound 3-piece molecule and the number of bound 5-piece molecule increased. It has been suggested that an excess of DCW3 resulted in an increase in the number of bound molecules in which DCW1-DCW3 were bound and in the number of bound molecules in which DCW3-DCW7 were bound. Based on these results, it was found that the binding efficiency of a plurality of fragments was improved after obtaining a reaction solution where the molar ratios of each bound fragment were the same.

[0128] [Example 7]

Linear double stranded DNA fragments of two or more types were linked using a RecA family recombinase protein and a 3'→5' exonuclease. In this reaction, the influence of the concentration of 3'→5'-exonuclease and the reaction time in the reaction solution was investigated.

[0129] DCW1-DCW7 were used as linked linear double-stranded DNA fragments. Wild-type E. coli RecA was used as the RecA family recombinase protein, and exonuclease III was used as the 3'→5'-exonuclease.

[0130] Specifically, reaction solutions were first prepared consisting of 1 nM each of DCW1-DCW7, 1 μM E. coli wild-type RecA, 20 mU/μl, 40 mU/μl, 80 mU/μl, 120 mU/μl, or 160 mU/µl exonuclease III, 20 mM Tris-HCl (pH 8.0), 4 mM DTT, 1 mM magnesium acetate, 100 µM ATP, 4 mM creatine phosphate, 20 ng/µl creatine kinase, 50 mM potassium glutamate, 150 mM TMAS, 5% by weight PEG8000 and 10% by volume DMSO. Then the reaction solutions were incubated at 42°C for 15 minutes, 30 minutes or 60 minutes to carry out the coupling reaction. 1 μl of the reaction solution after completion of the reaction was subjected to agarose gel electrophoresis, and the separated bands were stained with SYBR green.

[0131] The staining results are shown in FIG. 9. In this figure, "500 bp ladder" means the lane where the DNA ladder marker in Example 1 was used, and "In" means the lane where 1 μl of a solution containing 1 nM of each of DCW1-DCW7 is used. . As a result, in reaction solutions with 20 mU/µl, almost no bound molecule was formed even during an incubation time of 60 minutes. In contrast, in reaction solutions with 40 mU/µl of exonuclease III, almost no bound molecule was formed during the 30 minute incubation time, but a bound molecule of 7 fragments was formed during the 60 minute incubation time. In reaction solutions with 80-160 mU/µl of exonuclease III, a bound molecule of 7 fragments was formed even after an incubation time of 15 minutes. The reaction solution containing 80 mU/μl of exonuclease III and incubated for 30 minutes had the most bound molecule of 7 fragments and showed the best binding efficiency of multiple fragments.

[0132] [Example 8]

Linear double stranded DNA fragments of two or more types were linked using a RecA family recombinase protein and a 3'→5' exonuclease. In this reaction, the influence of the concentration of a linear double-stranded DNA fragment in the reaction solution was investigated.

[0133] DCW1-DCW49 (SEQ ID NO: 1 - SEQ ID NO: 49) were used as linked linear double-stranded DNA fragments. Similar to DCW1-DCW7, the DCW8-DCW49 regions extending from each end to 60 bases are homologous regions. Wild-type E. coli RecA was used as the RecA family recombinase protein, and exonuclease III was used as the 3'→5'-exonuclease.

[0134] Specifically, reaction solutions were first prepared consisting of 1 nM or 0.5 nM each of the linear double-stranded DNA fragments, 1 μM E. coli wild-type RecA, 80 mU/μl exonuclease III, 20 mM Trisa-HCl (pH 8.0), 4 mM DTT, 1 mM magnesium acetate, 100 μM ATP, 4 mM creatine phosphate, 20 ng/μl creatine kinase, 50 mM potassium glutamate, 150 mM TMAS, 5% by weight PEG8000 and 10% by volume DMSO. In a reaction solution containing 1 nM each of DCW1 - DCW20, the total amount of linear double-stranded DNA fragments is 20 nM (7.8 ng/µl). In a reaction solution containing 1 nM each of DCW1 - DCW25, the total amount of linear double-stranded DNA fragments is 25 nM (9.8 ng/µl). In a reaction solution containing 1 nM each of DCW1 - DCW30, the total amount of linear double-stranded DNA fragments is 30 nM (11.7 ng/µl). In a reaction solution containing 1 nM each of DCW1 - DCW40, the total amount of linear double-stranded DNA fragments is 40 nM (15.6 ng/µl). In a reaction solution containing 1 nM each of DCW1 - DCW49, the total amount of linear double-stranded DNA fragments is 49 nM (19.1 ng/µl). In a reaction solution containing 0.5 nM each of DCW1 - DCW49, the total amount of linear double-stranded DNA fragments is 24.5 nM (9.6 ng/µl). Then, these reaction solutions were incubated at 42°C for 30 minutes to carry out the coupling reaction. The reaction solution in the following volumes was subjected to agarose gel electrophoresis after completion of the reaction, and the separated bands were stained with SYBR green. The volume of the reaction solution prepared using 1 nM each of DCW1 to DCW20 was 1.25 µl; the volume of the reaction solution prepared using 1 nM each of DCW1 to DCW25 was 1 µl; the volume of the reaction solution prepared using 1 nM each of DCW1 to DCW30 was 0.83 µl; the volume of the reaction solution prepared using 1 nM each of DCW1 to DCW40 was 0.63 µl; the volume of the reaction solution prepared using 1 nM each of DCW1 to DCW49 was 0.51 µl; and the volume of the reaction solution prepared using 0.5 nM each of DCW1 to DCW49 was 1.02 µl.

[0135] The staining results are shown in FIG. 10. In reaction solutions containing 1 nM of each of the linear double-stranded DNA fragments, the formation of a bound molecule from a plurality of fragments is difficult because the number of contained fragments increases, that is, the total number of linear double-stranded DNA fragments in the reaction solution increases. Compared to reaction solutions containing DCW1 to DCW49, bound molecules that were associated with a larger number of fragments were formed in a reaction solution containing 0.5 nM each of the linear double-stranded DNA fragments, but not in a reaction solution containing 1 nM each of linear double-stranded DNA fragments. These results suggest that binding efficiency may be suppressed due to the very large total number of linear double-stranded DNA fragments in the reaction solution.

[0136] [Example 9]

Linear double stranded DNA fragments of two or more types were linked using a RecA family recombinase protein and a 3'→5' exonuclease. In this reaction, the influence of RecA family recombinase protein types in the reaction solution was investigated.

[0137] DCW1-DCW25 (SEQ ID NO: 1 - SEQ ID NO: 25) were used as linked linear double-stranded DNA fragments. Wild-type E. coli RecA or its F203W mutant was used as the RecA family recombinase protein, and exonuclease III was used as the 3'→5'-exonuclease.

[0138] Specifically, reaction solutions were first prepared consisting of 1 nM each of DCW1-DCW25, 0.5 μM, 0.75 μM, 1 μM, 1.25 μM, or 1.5 μM E. coli wild-type RecA, or its F203W mutant, 80 mU/µl exonuclease III, 20 mM Tris-HCl (pH 8.0), 4 mM DTT, 1 mM magnesium acetate, 100 µM ATP, 4 mM creatine phosphate, 20 ng/µl creatine kinase, 50 mM glutamate potassium, 150 mM TMAS, 5% by weight PEG8000 and 10% by volume DMSO. Then, the reaction solutions were incubated at 42°C for 30 minutes to carry out the coupling reaction, and then incubated at 65°C for 20 minutes. After incubation at 65°C and rapid cooling on ice, 1.5 μl of the reaction solution was subjected to agarose gel electrophoresis after completion of the reaction, and the separated bands were stained with SYBR green.

[0139] The staining results are shown in FIG. 11. In this figure, "500 bp ladder" means the lane where the DNA ladder marker in Example 1 was used, and "In" means the lane where 1.5 μl of a solution containing 1 nM of each of DCW1 is used. -DCW25. As a result, regardless of whether the RecA family recombinase protein is a wild-type protein or an F203W mutant, the number of coupled molecules produced from multiple fragments increased depending on the content of RecA. The reaction solution with the F203W mutant had more bound molecules produced from multiple fragments and had a higher binding efficiency than the reaction solution containing wild-type RecA.

[0140] [Example 10]

Linear double stranded DNA fragments of two or more types were linked using a RecA family recombinase protein and a 3'→5' exonuclease to form a linked circular molecule, and the linked circular molecule was subjected to RCR amplification.

[0141] First, a set of DCW1-DCW20 (SEQ ID NO: 1 - SEQ ID NO: 20) and Cm-oriC (DCW20) (SEQ ID NO: 50), which contained oriC and a pair of ter sequences built outside of oriC, respectively. The ter sequence is the sequence to which the Tus protein binds. The Tus protein functions to stop replication by a mechanism specific to the direction of the sequence. The term "embedding beyond oriC", when referring to the ter sequence, means that the ter sequence is inserted in such a way that, due to the action of a combination of proteins that bind to the ter sequence and inhibit replication, replication proceeds from oriC to oriC, stops . Cm-oriC (DCW20) is a linear 1298 bp double-stranded DNA fragment, and the 60 base region extending from base 1 to base 60 is a homologous region for binding to DCW20, which consists of the same base sequence , as is the 60-base sequence from bases 532 to 591 of DCW20. The 60 base region 1239 to 1298 Cm-Oric (DCW20) is the DCW1 binding region, which consists of the same base sequence as the 60 base sequence 1 to 60 DCW1. That is, if all fragments of DCW1 - DCW20 (21 fragments) and Cm-Oric (DCW20) were linked, then cDNA was obtained.

[0142] In addition, a set of DCW1-DCW25 (SEQ ID NO: 1 - SEQ ID NO: 25) and Cm-oriC (DCW25) (SEQ ID NO: 51), which included oriC . Cm-oriC (DCW25) is a linear 1298 bp double-stranded DNA fragment, and the 60 base region extending from base 1 to base 60 is a homologous region for binding to DCW25, which consists of the same base sequence , as is the 60-base sequence from bases 532 to 591 of DCW25. The 60 base region 1239 to 1298 Cm-Oric (DCW25) is the DCW1 binding region, which consists of the same base sequence as the 60 base sequence 1 to 60 DCW1. That is, if all 26 fragments of DCW1 - DCW25 and Cm-Oric (DCW25) were linked, then cDNA was obtained.

[0143] The E. coli F203W RecA mutant was used as the RecA family recombinase protein, and exonuclease III was used as the 3'→5' exonuclease. In addition, a mixed solution containing 60 nM Tus in the reaction mixture whose composition is shown in Table 1 was used as a reaction solution for RCR amplification. Tus was obtained and purified from an E. coli strain expressing Tus by a method including affinity column chromatography and column gel filtration.

[0144] [Table 1]

reaction mixture reaction buffer Tris-HCl (pH8.0) 20 mM Dithiothreitol 8 mM Potassium glutamate 150 mm Mg(OAc) ₂ 10 mM Creatine Phosphate 4 mM ATP 1 mM GTP, CTP, UTP 1 mM each dNTP 0.1 mM each tRNA 50 ng/µl NAD 0.25 mm Ammonium sulfate 10 mM Bovine Serum Albumin (BSA) 0.5 mg/ml Creatine kinase 20 ng/µl Enzymes SSB 400 nM IHF 20 nM DnaG 400 nM DnaN 40 nM Paul III* 5 nM DnaB, DnaC 20 nM DnaA 100 nM RNase H 10 nM Ligaz 50 nM Poli 50 nM GyrA, GyrB 50 nM Topo IV 5 nM Topo III 50 nM RecQ 50 nM

[0145] In Table 1, SSB means SSB derived from E. coli; IHF means a complex of IhfA and IhfB derived from E. coli; DnaG means DnaG derived from E. coli; DnaN means DnaN derived from E. coli; PolIII* denotes a DNA polymerase III* complex consisting of DnaX, HolA, HolB, HolC, HolD, DnaE, DnaQ and HolE derived from E. coli; DnaB means DnaB derived from E. coli; DnaC means DnaC derived from E. coli; DnaA means DnaA derived from E. coli; RNase H means RNase H derived from E. coli; ligase is a DNA ligase derived from E. coli; PolI means DNA polymerase I derived from E. coli; GyrA means GyrA derived from E. coli; GyrB means GyrB derived from E. coli; Topo IV means a complex of ParC and ParE derived from E. coli; Topo III means topoisomerase III derived from E. coli; and RecQ means RecQ derived from E. coli.

[0146] SSB was purified and obtained from an E. coli strain expressing SSB by a method including ammonium sulfate precipitation and ion exchange column chromatography.

IHF was obtained from E. coli strains co-expressing IhfA and IhfB by purification including ammonium sulfate precipitation and affinity column chromatography.

DnaG was obtained by purification from an E. coli strain expressing DnaG in steps that include ammonium sulfate precipitation, anion exchange column chromatography, and gel filtration column chromatography.

DnaN was purified and obtained from an E. coli strain expressing DnaN by a method including ammonium sulfate precipitation and anion exchange column chromatography.

PolIII* was generated and purified from E. coli strains co-expressing DnaX, HolA, HolB, HolC, HolD, DnaE, DnaQ, and HolE in steps including ammonium sulfate precipitation, affinity column chromatography, and gel filtration column chromatography.

DnaB and DnaC were generated and purified from E. coli strains co-expressing DnaB and DnaC in steps including ammonium sulfate precipitation, affinity column chromatography, and gel filtration column chromatography.

DnaA was generated and purified from a strain of Escherichia coli expressing DnaA in steps including ammonium sulfate precipitation, dialysis precipitation, and gel filtration column chromatography.

GyrA and GyrB were purified and prepared from a mixture of an E. coli strain expressing GyrA and an E. coli strain expressing GyrB by a method including ammonium sulfate precipitation, affinity column chromatography, and gel filtration column chromatography.

Topo IV was prepared from a mixture of a ParC-expressing Escherichia coli strain and a ParE-expressing Escherichia coli strain by a method including ammonium sulfate precipitation, affinity column chromatography, and gel filtration column chromatography.

Topo III was obtained from an E. coli strain expressing Topo III by purification by a method including ammonium sulfate precipitation and affinity column chromatography.

RecQ was obtained by purification from a strain of Escherichia coli expressing RecQ in steps including ammonium sulfate precipitation, affinity column chromatography, and gel filtration column chromatography.

RNase H, ligase and PolI were used as commercially available enzymes derived from E. coli (manufactured by Takara Bio Inc.).

[0147] In particular, reaction solutions were first prepared, consisting of a 2.5 nM or 5 nM set of linear double-stranded DNA fragments, 1 μM F203W RecA mutant, 80 mU/μl exonuclease III, 20 mM Trisa-HCl (pH 8.0) , 4 mM DTT, 1 mM magnesium acetate, 100 μM ATP, 4 mM creatine phosphate, 20 ng/μl creatine kinase, 50 mM potassium glutamate, 5 wt % PEG8000 and 10 v % DMSO. As a set of linear double-stranded DNA fragments, a set containing all equimolar amounts of DCW1 - DCW20 and Cm-oriC (DCW20), or a set containing all equimolar amounts of DCW1 - DCW25 and Cm-oriC (DCW25) was used. Then, these reaction solutions were incubated at 42°C for 30 minutes to carry out the coupling reaction, and then incubated at 65°C for 20 minutes for heat treatment, after which they were rapidly cooled on ice. After heat treatment and rapid cooling, 1 µl of reaction solutions containing 2.5 nM linear double-stranded DNA set and 0.5 µl of reaction solutions containing 5 nM linear double-stranded DNA set were subjected to agarose gel electrophoresis, and the separated bands were stained with SYBR green .

[0148] The staining results are shown in FIG. 12(a). In this figure, "1-20" denotes a lane where a solution containing a set of linear double-stranded DNA fragments with all equimolar amounts of DCW1 - DCW20 and Cm-oriC (DCW20) was used. In this figure, "1-25" denotes a lane where a solution containing a set of linear double-stranded DNA fragments with all equimolar amounts of DCW1 - DCW25 and Cm-oriC (DCW25) was used. As a result, it was confirmed that bound molecules of all sizes are contained in all reaction solutions.

[0149] Then, reaction mixtures were prepared by adding, after heat treatment and rapid cooling, 0.5 μl of the reaction solution to 4.5 μl of the RCR amplification reaction solution. The reaction mixtures were incubated at 30°C for 13 hours for RCR amplification. After completion of the reaction, 1 μl of the reaction mixture was subjected to agarose gel electrophoresis, and the separated bands were stained with SYBR green.

[0150] The staining results are shown in FIG. 12(b). In this figure, "1-20" and "1-25" mean the same tracks as in FIG. 12(a). As a result, in the lane of the RCR amplification product in the reaction solution, in which the set of bound DCW1 - DCW20 and Cm-oriC (DCW20) was present, a band of a supercoiled form of a circular bound molecule of 21 fragments (indicated in this figure as a "supercoil" of 21 fragments). In a lane of the RCR amplification product in a reaction solution containing a set of linked DCW1 - DCW25 and Cm-oriC (DCW25), a supercoiled 26-fragment ring-linked molecule band (denoted in this figure as a "supercoil" of 26 fragments) was detected. . Although after the coupling reaction, many bands were detected in the reaction solution (Fig. 12(a)), however, after RCR amplification, only a few bands were detected in the reaction mixture (Fig. 12(b)). It was confirmed that only circular linked molecules were amplified as a result of RCR amplification.

[0151] [Example 11]

Linear double stranded DNA fragments of two or more types were linked using a RecA family recombinase protein and a 3'→5' exonuclease to form a linked circular molecule, and the resulting linked circular molecule was subjected to RCR amplification. In this reaction, the effect of heat treatment and rapid cooling to RCR amplification was evaluated.

[0152] As linked linear double-stranded DNA fragments, a set of linear double-stranded DNA fragments with all equimolar amounts of DCW1 - DCW25 and Cm-oriC (DCW25) (referred to here as the "set of 20 nM linear double-stranded DNA fragments") of Example 10 was used. As a protein RecA family recombinases used the E. coli F203W RecA mutant, and exonuclease III was used as the 3'→5'-exonuclease. In addition, as a reaction solution for RCR amplification, a mixed solution containing 60 nM Tus in the reaction mixture, the composition of which is shown in Table 1, was used.

[0153] In particular, reaction solutions were first prepared consisting of a 20 nM set of linear double-stranded DNA fragments, 1.5 μM F203W RecA mutant, 80 mU/μl exonuclease III, 20 mM Tris-HCl (pH 8.0), 4 mM DTT, 1 mM magnesium acetate, 100 μM ATP, 4 mM creatine phosphate, 20 ng/μl creatine kinase, 50 mM potassium glutamate, 150 mM TMAS, 5% by weight PEG8000 and 10% by volume DMSO. Then, these reaction solutions were incubated at 42°C for 30 minutes to carry out the coupling reaction, and then incubated at 50°C or 65°C for 2 minutes for heat treatment, after which they were quickly cooled on ice. After rapid cooling, 1.5 μl of the reaction solutions were subjected to agarose gel electrophoresis and the separated bands were stained with SYBR green.

[0154] The staining results are shown in FIG. 13(a). In this figure, "500 bp ladder" means the lane where the DNA ladder marker in Example 1 was used, and "In" means the lane where 1.5 µl of a solution containing a 20 nM set of linear double-stranded DNA fragments is used. . "-" means a lane where the reaction solution was not subjected to heat treatment after the coupling reaction. "50°C" corresponds to the lane where the reaction solution was subjected to heat treatment at 50°C for 2 minutes. "65°C" corresponds to the lane where the reaction solution was subjected to heat treatment at 65°C for 2 minutes. As shown in FIG. 13(a), in the reaction solutions without heat treatment, DNA did not migrate to form a blurred band, while in the reaction solutions subjected to heat treatment, most of the smeared bands were removed.

[0155] Then, the reaction mixtures were prepared by adding, after heat treatment and rapid cooling, 0.5 μl of the reaction solution to 4.5 μl of the RCR amplification reaction solution. The reaction mixtures were incubated at 30°C for 13 hours for RCR amplification. As a control, 0.5 µl of the reaction solution, after heat treatment at 65°C and rapid cooling, was added to 4.5 µl of a TE solution consisting of 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA to obtain pre-amplification solution. After completion of the reaction, 1 μl of the pre-amplification solution and the reaction mixture were subjected to agarose gel electrophoresis, and the separated bands were stained with SYBR green.

[0156] The staining results are shown in FIG. 13(b). In this figure, "MK3" means the lane where the DNA ladder marker was used. As a result, in the reaction mixture obtained by RCR amplification of the reaction solution, where the ring-linked molecules were formed by the coupling reaction, a band of the supercoiled form of the 26-fragment ring-linked molecule (indicated as “25-fragment scDNA” in this figure) was detected( "-", "50°C", "65°C" in Fig. 13(b)). In the pre-amplification solution, no band was detected ("Entrance" in Fig. 13(b)). Two broad bands, where the migration distance was greater than for the supercoiled bonded molecule band of 26 fragments, were detected in the reaction mixture without heat treatment ("-"), while in the reaction mixture subjected to heat treatment at 50°C ("50°C" ), these bands were thin, and were not detected at all when heat treated at 65°C ("65°C"). Based on these results, it was found that the DNA bands where the migration distance was greater than the 26-fragment supercoiled linked molecule band corresponded to amplification products of circular linked molecules obtained by non-specific binding, and such non-specific amplification products can be inhibited by heat treatment and rapid cooling to RCR amplification.

[0157] [Example 12]

Linear double-stranded DNA fragments of 26 or 36 types were linked using RecA family recombinase protein and 3'→5'-exonuclease to form a linked circular molecule, and the resulting linked circular molecule was subjected to RCR amplification.

[0158] A set of DCW1-DCW25 (SEQ ID NO: 1 - SEQ ID NO: 25) and Km-oriC (DCW25) (SEQ ID NO: 52) was used as linked linear double-stranded DNA fragments, which included oriC. Km-oriC (DCW25) is a linear 1509 bp double-stranded DNA fragment, and the 60 base region extending from the first to the 60th base is a homologous region for binding to DCW25, which consists of the same base sequence , as is the 60-base sequence from bases 532 to 591 of DCW25. The 60 base region from bases 1450 to 1509 of Km-oriC (DCW25) is the DCW1 binding region, which consists of the same base sequence as the 60 base sequence from bases 1 to 60 of DCW1 . That is, cDNA was obtained by linking all 26 types of DCW1 fragments - DCW25 and Km-oriC (DCW25).

[0159] A set of DCW1-DCW35 (SEQ ID NO: 1 - SEQ ID NO: 35) and Km-oriC (DCW35) (SEQ ID NO: 53) was used as linked linear double-stranded DNA fragments, which included oriC and a pair of sequences ter built outside of oriC, respectively. Km-oriC (DCW35) is a linear 1509 bp double-stranded DNA fragment, and the 60 base region extending from the first to the 60th base is a homologous region for binding to DCW35, which consists of the same base sequence as is the 60-base sequence from bases 532 to 591 of DCW35. The 60 base region from bases 1450 to 1509 of Km-oriC (DCW35) is the region for binding to DCW1, which consists of the same base sequence as the 60 base sequence from bases 1 to 60 DCW1. That is, cDNA was obtained by linking all 36 types of DCW1 fragments - DCW35 and Km-oriC (DCW35).

[0160] E. coli F203W RecA mutant was used as the RecA family recombinase protein, and exonuclease III was used as the 3'→5' exonuclease. In addition, as a reaction solution for RCR amplification, a mixed solution containing 60 nM Tus in the reaction mixture, the composition of which is shown in Table 1, was used.

[0161] Specifically, reaction solutions were first prepared consisting of a 20 nM set of linear double-stranded DNA fragments, 1.5 μM F203W RecA mutant, 80 mU/μl exonuclease III, 20 mM Tris-HCl (pH 8.0), 4 mM DTT, 1 mM magnesium acetate, 100 μM ATP, 4 mM creatine phosphate, 20 ng/μl creatine kinase, 50 mM potassium glutamate, 150 mM TMAS, 5% by weight PEG8000 and 10% by volume DMSO. As a set of linear double-stranded DNA fragments, a set containing all equimolar amounts of DCW1 - DCW25 and Cm-oriC (DCW25) or a set containing all equimolar amounts of DCW1 - DCW35 and Cm-oriC (DCW35) was used. Then, these reaction solutions were incubated at 42°C for 30 minutes to carry out the coupling reaction, and then incubated at 65°C for 5 minutes for heat treatment, after which they were quickly cooled on ice. After heat treatment and rapid cooling, 1.5 μl of the reaction solutions were subjected to agarose gel electrophoresis, and the separated bands were stained with SYBR green.

[0162] The staining results are shown in FIG. 14(a). In this figure, "Input" means the lane where 1.5 μl of a solution containing a set of 20 nM linear double-stranded DNA fragments is used. "DCW1-25 Km-oriC" means a lane where a reaction solution containing a set with all equimolar amounts of DCW1 - DCW25 and Km-oriC (DCW25) is used. "DCW1-35 Km-oriC" means a lane where a reaction solution containing a set with all equimolar amounts of DCW1 - DCW35 and Km-oriC (DCW35) is used. As shown in FIG. 14(a), using any set of linear double-stranded DNA fragments, bound molecules from the plurality of fragments were generated by a coupling reaction.

[0163] Then, 0.5 µl of the reaction solution, after heat treatment and rapid cooling, was added to 4.5 µl of the RCR amplification reaction solution to obtain a reaction mixture. The RCR amplification reaction was carried out by incubating the reaction mixture at 30° C. for 16 hours. Then, 0.5 μl of each product of the RCR amplification reaction was diluted to 4.5 μl with the reaction buffer, which was obtained by removing only the group of enzymes from the reaction mixture shown in Table 1, and then again incubated at 30°C for 30 minutes. The re-incubation treatment after dilution stimulates the reaction of replication extension and separation of the amplification intermediate in said product and increases the amount of supercoiled DNA produced, which is the final product. 0.5 µl of the reaction solution in which the coupling reaction was carried out using DCW1 to DCW25 followed by heat treatment and rapid cooling was added to 4.5 µl of a TE solution consisting of 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA . Thus, this resulting solution was used as a control. This resulting solution was used as a control to prepare a pre-amplification solution. After re-incubation, 2.5 μl of the pre-amplification solution and the reaction mixture were subjected to agarose gel electrophoresis, and the separated bands were stained with SYBR green.

[0164] The staining results are shown in FIG. 14(b). As a result, in the reaction mixture obtained by RCR amplification after binding a set of linear double-stranded DNA fragments containing 26 fragments, a band of a supercoiled form of a circular linked molecule of 26 fragments (indicated in this figure as "26 fragments scDNA") was detected (" DCW1-25 Km-oriC" in Fig. 13(b)). In the reaction mixture obtained by RCR amplification after binding a set of linear double-stranded DNA fragments containing 36 fragments, a band of a supercoiled form of a circular linked molecule of 36 fragments (indicated in this figure as “36 fragments scDNA”) (“DCW1-35 Km-oriC" in Fig. 13(b)). In the pre-amplification solution, no band was detected ("Entrance" in Fig. 14(b)). Based on these results, it was confirmed that a multi-fragment circular-linked molecule, namely 36 fragments, can be obtained by the method of the invention, and this circular-linked molecule can be amplified by RCR amplification. However, the 36-fragment bound molecule, in contrast to the 26-fragment bound molecule, had a greater number of non-specific amplification products obtained by RCR amplification.

[0165] [Example 13]

The "NEBuilder HiFi for DNA assembly" kit (manufactured by NEB) used in the Gibson assembly method of linking a plurality of double-stranded DNA fragments (Patent Document 3) is commercially available. In this kit, linear double-stranded DNA fragments of two or more types, having a homologous region of 15-20 bases at the end, were linked by the NEB method, that is, by adding the DNA fragments to a mixed solution (Master Mix) followed by incubation of the mixed solution at 50° C for 15-60 minutes. The mixed solution was supplied with the kit and contained 5'→3' exonuclease, DNA polymerase and DNA ligase.

[0166] The binding efficiency in the method of obtaining DNA according to the invention, where the binding reaction was carried out using the RecA family recombinase protein and 3'→5'-exonuclease, was comparable to the efficiency in the NEB method.

[0167] A set containing all equimolar amounts of DCW1 - DCW25 (SEQ ID NO:1-SEQ ID NO:25) and Km-oriC (DCW25) was used as linked linear double-stranded DNA fragments, which was used in Example 12. As The F203W RecA mutant of E. coli was used for the RecA family recombinase protein, and exonuclease III was used as the 3'→5'-exonuclease. In addition, as a reaction solution for RCR amplification, a mixed solution containing 60 nM Tus in the reaction mixture, the composition of which is shown in Table 1, was used.

[0168] Specifically, reaction solutions were first prepared consisting of a 20 nM or 60 nM set of linear double-stranded DNA fragments, 1.5 μM F203W RecA mutant, 80 mU/μl exonuclease III, 20 mM Trisa-HCl (pH 8.0) , 4 mM DTT, 1 mM magnesium acetate, 100 μM ATP, 4 mM creatine phosphate, 20 ng/μl creatine kinase, 50 mM potassium glutamate, 150 mM TMAS, 5 wt% PEG8000 and 10 v/v DMSO, for their use in the method according to the invention (in the RA method). Then, these reaction solutions were incubated at 42°C for 30 minutes to carry out the coupling reaction, and then incubated at 65°C for 5 minutes for heat treatment, after which they were quickly cooled on ice. After rapid cooling, 1.5 μl of the reaction solutions were subjected to agarose gel electrophoresis and the separated bands were stained with SYBR green.

[0169] In the NEB method, a reaction solution was prepared by mixing a 20 nM or 60 nM set of linear double-stranded DNA fragments with a solution 2-fold diluted with the “Master Mix” supplied with the specified set. Then, the reaction solution was incubated at 50°C for 60 minutes to carry out the coupling reaction. After the binding reaction, 1.5 μl of the reaction solution was subjected to agarose gel electrophoresis, and the separated bands were stained with SYBR green.

[0170] The staining results are shown in FIG. 15(a). In this figure, "Input" means the lane where 1.5 μl of a solution containing a set of 20 nM linear double-stranded DNA fragments is used. "RA" means the lane where the reaction solution obtained by the method according to the invention (RA method) is used. "NEB" means the lane where the reaction solution obtained by the NEB method is used. As shown in FIG. 15(a), a significant number of fragments were obtained by carrying out the coupling reaction by the RA method, regardless of whether the set of linear double-stranded DNA fragments in the reaction solution had a concentration of 20 nM or 60 nM. On the other hand, in the reaction solution where the coupling reaction is carried out by the NEB method, only bound molecules of 2-3 fragments were obtained.

[0171] Then, the reaction mixtures were prepared, after heat treatment and rapid cooling, by adding 0.5 μl of the reaction solution to 4.5 μl of the RCR amplification reaction solution. The reaction mixtures were incubated at 30°C for 16 hours for RCR amplification. Then, 0.5 μl of each product of the RCR amplification reaction was diluted to 4.5 μl with the reaction buffer, which was obtained by removing only the group of enzymes from the reaction mixture shown in Table 1, and then again incubated at 30°C for 30 minutes. After re-incubation, 2.5 μl of the reaction mixture was subjected to agarose gel electrophoresis and the separated bands were stained with SYBR green.

[0172] The staining results are shown in FIG. 15(b). As a result, in the reaction mixture obtained by RCR amplification after the coupling reaction by the method according to the invention (RA method) (in this figure "RA"), a band of supercoiled form of a circular bonded molecule was detected, where all 26 fragments were associated (in this figure , "supercoil of 25 fragments"). On the other hand, in the reaction mixture obtained by RCR amplification after the NEB binding reaction) (NEB in this figure), the 26-fragment ring-linked molecule band was not detected, and 26 fragments could not be bound by the NEB method. In both reaction mixtures, products that become concatemers due to non-specific rolling-ring replication and circular products of DNA multimers (catenans) that remain unseparated after replication were also detected with separation limitation by agarose gel electrophoresis ( on this figure, "multimer").

[0173] [Example 14]

Long genomic fragments were linked to each other to form a circular linked molecule, and the circular linked molecule was subjected to RCR amplification.

[0174] The Xba I digest (15 fragments, DGF-298/XbaI) of E. coli strain genomic DNA (DGF-298WΔ100::revΔ234::SC) was used as the long chain genomic fragment. Of these hydrolysates, a 325 kb genomic fragment. (325k genomic fragment) and 220 kb genomic fragment. (220k genomic fragment) were linked to each other via a binding fragment containing oriC (Cm-oriC fragment) to obtain a product in ring form. 1298 bp was used as a binding fragment for cyclization of the 325k genomic fragment. a linear double-stranded DNA fragment including oriC (Cm-oriC/325k fragment, SEQ ID NO:59). The upstream end of this binding fragment was homologous to the downstream end of the 325k genomic fragment (i.e., the 60 bases at the upstream end of this fragment consisted of the same base sequence as the 60 bases at the downstream end), and the downstream end of this binding fragment was homologous to the upstream end of the 325k genomic fragment (i.e., the 60 bases located at the downstream end of this fragment consisted of the same base sequence as the 60 bases at the upstream end). 1298 bp was used as a binding fragment for cyclization of the 220k genomic fragment. a linear double-stranded DNA fragment including oriC (Cm-oriC/220k fragment, SEQ ID NO:60). The upstream end of this binding fragment was homologous to the downstream end of the 220k genome fragment (i.e., the 60 bases at the upstream end of this fragment consisted of the same base sequence as the 60 bases at the downstream end), and the downstream end the region of this binding fragment was homologous to the upstream end of the 220k genomic fragment (i.e., the 60 bases located at the downstream end of this fragment consisted of the same base sequence as the 60 bases at the upstream end). In addition, the reaction mixture whose composition is shown in Table 1 was used as a solution for the RCR amplification reaction.

[0175] Specifically, an Xba I digest of E. coli genomic DNA (DGF-298/XbaI, 4.8 ng/µl) and a Cm-oriC/325k fragment having a region homologous to that of the 325k genomic fragment that represents genomic target fragment (240 pM) was added to the reaction mixture RA [20 mM Tris-HCl (pH 8.0), 4 mM DTT, 150 mM KOAc, 10 mM Mg(OAc) ₂ , 100 μM ATP, 5% by weight PEG8000, 40 mU/µl exonuclease III, 1 µM RecA F203W mutant E. coli] (5 µl), and the solution was incubated at 30°C for 60 minutes to carry out the binding reaction. 0.5 μl of the resulting RA product was added to the RCR amplification reaction solution (4.5 μl), and the amplification reaction was carried out using a thermal cycle (1 cycle at 37°C for 1 minute, and then at 24°C for 30 minutes, 40 cycles in total). The binding reaction was carried out in a similar manner, but using a Cm-oriC/220k fragment instead of a Cm-oriC/325k fragment, and incorporating a 220 kb target genomic fragment, followed by an RCR amplification reaction. As a control, a 200 kb circular oriC plasmid. subjected to RCR amplification. 50 μM diethylenetriaminepentaacetic acid was added to the RCR amplification reaction solution of the binding product of the 325k genomic fragment to stabilize long-stranded DNA.

[0176] After the reaction, 1 μl of the reaction mixture was subjected to agarose gel electrophoresis, and the separated bands were stained with SYBR green. The staining results are shown in Fig. 16. On this figure "220 kbp." means a lane where an amplification product obtained by reaction with a 220 kb target genomic fragment is used. "325 kbp" means a lane where an amplification product obtained by reaction with a 325 kb target genomic fragment is used. “200 kbp (RCR control)" means the lane where the product obtained by direct amplification of the 200 kb circular oriC plasmid is used. As a result, the supercoiled form of a 220 kbp circular bonded molecule. and a supercoiled form of a 325 kb circular bonded molecule. were detected by reaction with a 220 kb target genomic fragment. and by reaction with a 325 kb target genomic fragment, respectively. Based on these results, it was found that a double-stranded DNA fragment of at least 325 kb. can be cyclized by the method of obtaining DNA according to the invention.

[0177] [Example 15]

Linear double stranded DNA fragments of two or more types were linked using a RecA family recombinase protein and a 3'→5' exonuclease. In this reaction, the effect of the concentration of creatine phosphate in a reaction solution containing an ATP-regenerating system consisting of creatine kinase and creatine phosphate was investigated.

[0178] DCW1-DCW10 (SEQ ID NO: 1 - SEQ ID NO: 10) were used as linked linear double-stranded DNA fragments. The F203W RecA mutant of E. coli was used as the RecA family recombinase protein, and exonuclease III was used as the 3'→5'-exonuclease.

[0179] In particular, reaction solutions were first prepared consisting of 2 nM each of DCW1-DCW10, 1.5 μM F203W RecA mutant, 80 mU/μl exonuclease III, 20 mM Tris-HCl (pH 8.0), 4 mM DTT, 1 mM magnesium acetate, 50 mM potassium glutamate, 100 µM ATP, 150 mM TMAS, 5 wt% PEG8000, 10 v/v DMSO, 20 ng/µl creatine kinase and 0 mM (no addition), 0.1 mM, 0.4 mM, 1 mM, 4 mM or 10 mM creatine phosphate. Then, these reaction solutions were incubated at 42°C for 30 minutes to carry out the coupling reaction, and then incubated at 65°C for 2 minutes for heat treatment, after which they were quickly cooled on ice. After heat treatment and rapid cooling, 1.5 μl of the reaction solutions were subjected to agarose gel electrophoresis, and the separated bands were stained with SYBR green.

[0180] The staining results are shown in FIG. 17. In this figure, "Input" means the lane where 2 µl of a solution containing 2 nM of each of DCW1-DCW10 is used. As a result, for the samples subjected to the binding reaction, a band of the associated molecule was detected, obtained by binding fragments of all 10 types in the samples with 0.4-10 mm creatine phosphate. Specifically, samples containing 1 mM or 4 mM creatine phosphate were found to have a large number of 10 fragment bound molecules, and among these samples, a sample containing 4 mM creatine phosphate had a large number of 2-9 fragment bound molecules. , and showed excellent binding efficiency.

[0181] [Example 16]

Linear double stranded DNA fragments of two or more types were linked using a RecA family recombinase protein and a 3'→5' exonuclease. In this reaction, the effect of creatine kinase concentration in a reaction solution containing an ATP-regenerating system consisting of creatine kinase and creatine phosphate was investigated.

[0182] DCW1-DCW10 (SEQ ID NO: 1 - SEQ ID NO: 10) were used as linked linear double-stranded DNA fragments. The F203W RecA mutant of E. coli was used as the RecA family recombinase protein, and exonuclease III was used as the 3'→5'-exonuclease.

[0183] In particular, reaction solutions were first prepared consisting of 2 nM each of DCW1-DCW10, 1.5 μM F203W RecA mutant, 80 mU/μl exonuclease III, 20 mM Tris-HCl (pH 8.0), 4 mM DTT, 1 mM magnesium acetate, 50 mM potassium glutamate, 100 µM ATP, 150 mM TMAS, 5 wt% PEG8000, 10 v/v DMSO, 4 mM creatine phosphate and 0 ng/µl, 20 ng/µl, 50 ng/ µl or 200 ng/µl creatine kinase. Then, these reaction solutions were incubated at 42°C for 30 minutes to carry out the coupling reaction, and then incubated at 65°C for 2 minutes for heat treatment, after which they were quickly cooled on ice. After heat treatment and rapid cooling, 1.5 μl of the reaction solutions were subjected to agarose gel electrophoresis, and the separated bands were stained with SYBR green.

[0184] The staining results are shown in FIG. 18. In this figure, "Input" means the lane where 2 µl of a solution containing 2 nM of each of DCW1-DCW10 is used. "Buffer" means the lane where the sample is used, which did not add creatine kinase (0 ng/μl). As a result, for the samples subjected to the binding reaction, a band of the associated molecule was detected, obtained by binding fragments of all 10 types in all samples with the addition of creatine kinase.

[0185] [Example 17]

Linear double stranded DNA fragments of two or more types were linked using a RecA family recombinase protein and a 3'→5' exonuclease. In this reaction, the influence of the ATP-regenerating system consisting of pyruvate kinase and phosphoenol pyruvate was investigated.

[0186] A set of DCW1-DCW35 (SEQ ID NO: 1 - SEQ ID NO: 35) and Km-oriC (DCW35) (SEQ ID NO: 53) was used as linked linear double-stranded DNA fragments, which included oriC and a pair of sequences ter built outside of oriC, respectively. The F203W RecA mutant of E. coli was used as the RecA family recombinase protein, and exonuclease III was used as the 3'→5'-exonuclease.

[0187] Specifically, reaction solutions were first prepared consisting of 0.6 nM each of DCW1-DCW35 (SEQ ID NO: - SEQ ID NO: 5) and Km-oriC (DCW35) (SEQ ID NO: 53), 1 .5 μM F203W RecA mutant, 80 mU/μl exonuclease III, 20 mM Tris-HCl (pH 8.0), 4 mM DTT, 1 mM magnesium acetate, 50 mM potassium glutamate, 100 μM ATP, 150 mM TMAS, 5% by weight PEG8000, 10% v/v DMSO, 2 mM phosphoenol pyruvate, and 10 ng/µl, 32 ng/µl, or 100 ng/µl pyruvate kinase. A comparison reaction solution was prepared in a similar manner, except that 2 mM creatine phosphate was used instead of 2 mM phosphoenol pyruvate, and 20 ng/μl of creatine kinase as a mixture was used instead of pyruvate kinase. The reference reaction solution was also prepared in a similar manner, except that phosphoenol pyruvate and pyruvate kinase were not used. Then, these reaction solutions were incubated at 42°C for 30 minutes to carry out the coupling reaction, and then incubated at 65°C for 2 minutes for heat treatment, after which they were rapidly cooled on ice. After heat treatment and rapid cooling, 1.5 μl of the reaction solutions were subjected to agarose gel electrophoresis, and the separated bands were stained with SYBR green.

[0188] The staining results are shown in FIG. 19. In this figure, "Input" means a lane where 2 μl of a solution containing 0.6 nM each of DCW1-DCW35 (SEQ ID NO: 1 - SEQ ID NO: 35) and Km-oriC (DCW35) is used. "ATP regeneration" means a lane where a sample is used without pyruvate phosphoenol and pyruvate kinase. "2 mM CP, 20 ng/µl CK" means a lane where a sample containing creatine phosphate and creatine kinase is used. "2 mM PEP" means a lane where a sample containing phosphoenol pyruvate and pyruvate kinase is used. As a result, bands of bound molecules from multiple fragments were detected in samples containing an ATP-regenerating system consisting of 2 mM phosphoenol pyruvate and 100 ng/μl pyruvate kinase, similar to samples containing an ATP-regenerating system consisting of creatine phosphate and creatine kinase. .

[0189] [Example 18]

Linear double stranded DNA fragments of two or more types were linked using a RecA family recombinase protein and a 3'→5' exonuclease. In this reaction, the effect of the ATP-regenerating system consisting of polyphosphate kinase and polyphosphate was investigated.

[0182] DCW1-DCW10 (SEQ ID NO: 1 - SEQ ID NO: 10) were used as linked linear double-stranded DNA fragments. The F203W RecA mutant of E. coli was used as the RecA family recombinase protein, and exonuclease III was used as the 3'→5'-exonuclease.

[0183] Specifically, reaction solutions were first prepared consisting of 2 nM each of DCW1-DCW10, 1 μM wild-type RecA, 80 mU/μl exonuclease III, 20 mM Tris-HCl (pH 8.0), 4 mM DTT, 1 mM magnesium acetate, 50 mM potassium glutamate, 100 µM ATP, 150 mM TMAS, 5% w/w PEG8000, 10% v/v DMSO, 1 mM, 4 mM or 10 mM polyphosphate and 20 ng/µl, 60 ng/µl or 150 ng/µl polyphosphate kinase. Then, these reaction solutions were incubated at 42°C for 30 minutes to carry out the coupling reaction, and then incubated at 65°C for 2 minutes for heat treatment, after which they were rapidly cooled on ice. After heat treatment and rapid cooling, 1.5 μl of the reaction solutions were subjected to agarose gel electrophoresis, and the separated bands were stained with SYBR green.

[0184] The staining results are shown in FIG. 20. In this figure, "Input" means the lane where 2 μl of a solution containing 2 nM of each of DCW1-DCW10 is used. As a result, for the samples subjected to the binding reaction, a band of a bound molecule was detected, obtained by binding fragments of all 10 types in a sample containing 60 ng/μl of polyphosphate kinase and 1 mm of polyphosphate.

[0193] [Example 19]

Linear double stranded DNA fragments of two or more types were linked using a RecA family recombinase protein and a 3'→5' exonuclease. In this reaction, the effect of using a combination of 3'→5'-exonuclease specific for linear double-stranded DNA and 3'→5'-exonuclease specific for single-stranded DNA was investigated.

[0194] DCW1-DCW10 (SEQ ID NO: 1 - SEQ ID NO: 10) were used as linked linear double-stranded DNA fragments. Exonuclease III and exonuclease I were used as 3'→5'-exonuclease specific for linear double-stranded DNA and 3'→5'-exonuclease specific for single-stranded DNA, respectively.

[0195] In particular, reaction solutions were first prepared consisting of 1 nM each of DCW1-DCW10, 1 μM wild-type RecA, 80 mU/μl exonuclease III, 20 mM Tris-HCl (pH 8.0), 4 mM DTT, 1 mM magnesium acetate, 50 mM potassium glutamate, 100 µM ATP, 150 mM TMAS, 5% w/w PEG8000, 10% v/v DMSO, 1 mM, 4 mM or 10 mM polyphosphate and 20 ng/µl, 60 ng/µl or 150 ng/µl polyphosphate kinase. Then, these reaction solutions were incubated at 42°C for 30 minutes to carry out the coupling reaction, and then incubated at 65°C for 2 minutes for heat treatment, after which they were rapidly cooled on ice. After heat treatment and rapid cooling, 1.5 μl of the reaction solutions were subjected to agarose gel electrophoresis, and the separated bands were stained with SYBR green.

[0196] The staining results are shown in FIG. 21. In this figure, "Input" means the lane where 2 µl of a solution containing 1 nM of each of DCW1-DCW10 is used. As a result, in all samples subjected to the binding reaction, a band of the bound molecule obtained by binding fragments of all 10 types was detected. The amount of bound molecule obtained by binding fragments of all 10 types increased depending on the content of exonuclease I. Based on these results, it was found that the addition of exonuclease I stimulates the binding reaction using exonuclease III and RecA.

[0197] [Example 20]

Linear double-stranded DNA fragments of 36 types were linked using the RecA family recombinase protein; 3'→5'-exonuclease specific for linear double-stranded DNA; and a 3'→5' exonuclease specific for single stranded DNA to form a linked circular molecule, and this linked circular molecule was subjected to RCR amplification.

[0198] A set of DCW1-DCW35 (SEQ ID NO: 1 - SEQ ID NO: 35) and Km-oriC (DCW35) (SEQ ID NO: 52) was used as linked linear double-stranded DNA fragments, which included oriC and a pair of sequences ter built outside of oriC, respectively. Wild-type E. coli RecA was used as the RecA family recombinase protein. Exonuclease III and exonuclease I were used as 3'→5'-exonuclease specific for linear double-stranded DNA and 3'→5'-exonuclease specific for single-stranded DNA, respectively. In addition, as a reaction solution for RCR amplification, a mixed solution containing 60 nM Tus in the reaction mixture, the composition of which is shown in Table 1, was used.

[0199] In particular, reaction solutions were first prepared consisting of 0.6 nM each of DCW1-DCW35 and Km-oriC (DCW35), 1.5 μM wild-type RecA, 80 mU/μl exonuclease III, 20 mM Trisa-HCl (pH 8.0), 4 mM DTT, 1 mM magnesium acetate, 50 mM potassium glutamate, 100 μM ATP, 150 mM TMAS, 5% w/w PEG8000, 10% v/v DMSO, 20 ng/μl creatine kinase, 4 mM phosphate creatine and 0 U/µl (no addition), 0.3 U/µl, or 1 U/µl exonuclease I. Then, these reaction solutions were incubated at 42°C for 30 minutes to carry out the binding reaction, and then incubated at 65°C for 2 minutes for heat treatment, and then quickly cooled on ice. After heat treatment and rapid cooling, 1.5 μl of the reaction solutions were subjected to agarose gel electrophoresis and the separated bands were stained with SYBR green.

[0200] The staining results are shown in FIG. 22(a). In this figure, "Entrance" means the lane where 2 μl of a solution containing 0.6 nM each of DCW1-DCW35 and Km-oriC (DCW35) is used. As shown in FIG. 22(a), bound molecules from multiple fragments were detected in all samples. A sample to which more exonuclease I has been added has more bound molecules from multiple fragments.

[0201] Then, reaction mixtures were prepared, after heat treatment and rapid cooling, by adding 0.5 μl of the reaction solution to 4.5 μl of the RCR amplification reaction solution. The reaction mixtures were incubated at 30°C for 16 hours for RCR amplification. Then, 0.5 μl of each product of the RCR amplification reaction was diluted to 4 μl with the reaction buffer, which was obtained by removing only the group of enzymes from the reaction mixture shown in Table 1, and then again incubated at 30°C for 30 minutes. After re-incubation, 2.5 μl of the reaction mixture was subjected to agarose gel electrophoresis and the separated bands were stained with SYBR green.

[0202] The staining results are shown in FIG. 22(b). As a result, in the sample containing exonuclease I, a band of a supercoiled form of a circular linked 36-fragment molecule (indicated as “36-fragment scDNA” in this figure) (“36-fragment supercoil” in Fig. 13(b)) was detected. On the other hand, in the sample without the addition of exonuclease I, this band was not observed. Based on these results, it was found that the efficiency of the coupling reaction carried out using RecA and exonuclease III was stimulated after the addition of exonuclease I, resulting in a circular linked molecule obtained by binding fragments of all 36 types.

[0203] [Example 21]

Linear double-stranded DNA fragments of 50 types were linked using a RecA family recombinase protein; 3'→5'-exonucleases; and a 3'→5' exonuclease specific for single stranded DNA to form a linked circular molecule, and this linked circular molecule was subjected to RCR amplification.

[0204] A set of DCW1-DCW49 (SEQ ID NO: 1 - SEQ ID NO: 49) and Km-oriC (DCW49) (SEQ ID NO: 62) was used as linked linear double-stranded DNA fragments, which included oriC and a pair of sequences ter built outside of oriC, respectively. Km-oriC (DCW49) is a linear 1509 bp double-stranded DNA fragment, and the 60 base region extending from the first to the 60th base is a homologous region for binding to DCW49, which consists of the same base sequence , as is the 60-base sequence from bases 532 to 591 of DCW49. The 60 base region from bases 1450 to 1509 of Km-oriC (DCW49) is the region for binding to DCW1, which consists of the same base sequence as the 60 base sequence from bases 1 to 60 DCW1. That is, cDNA was obtained by linking all 50 types of DCW1 fragments - DCW49 and Km-oriC (DCW49).

[0205] As a positive control, a set of DCW1-DCW35 (SEQ ID NO: 1 - SEQ ID NO: 35) and Km-oriC (DCW35) was used, which included oriC and a pair of ter sequences inserted outside of oriC, respectively. Wild-type E. coli RecA was used as the RecA family recombinase protein. Exonuclease III and exonuclease I were used as the 3'→5'-exonuclease specific for linear double-stranded DNA and 3'→5'-exonuclease specific for single-stranded DNA. solution containing 60 nM Tus in the reaction mixture, the composition of which is given in Table 1.

[0206] In particular, reaction solutions were first prepared consisting of 0.6 nM each of DCW1-DCW49 and Km-oriC (DCW49), 1.5 μM wild-type RecA, 80 mU/μl exonuclease III, 20 mM Trisa-HCl (pH 8.0), 4 mM DTT, 1 mM magnesium acetate, 50 mM potassium glutamate, 100 μM ATP, 150 mM TMAS, 5% w/w PEG8000, 10% v/v DMSO, 20 ng/μl creatine kinase, 4 mM phosphate creatine and 0.3 U/µl exonuclease I. The reaction solution was prepared in a similar manner, except that instead of 0.6 nM each of DCW1-DCW49 and Km-oriC (DCW49), a mixture of 0.6 nM each of DCW1- DCW35 and Km-oriC (DCW35). Then, these reaction solutions were incubated at 42°C for 30 minutes to carry out the coupling reaction, and then incubated at 65°C for 2 minutes for heat treatment, after which they were rapidly cooled on ice. After heat treatment and rapid cooling, 1.5 μl of the reaction solutions were subjected to agarose gel electrophoresis, and the separated bands were stained with SYBR green.

[0207] The staining results are shown in FIG. 23(a). In this figure, for "20 nM (8.8 ng/mL) DCW1-35 Km-oriC", "Input" means the lane where 1.5 µl of a solution containing 0.6 nM each of DCW1-DCW35 and Km -oriC (DCW35) and "RA" means the lane where a reaction solution containing 0.6 nM each of DCW1-DCW35 and Km-oriC (DCW35) is used. For "30 nM (12.1 ng/mL) DCW1-49 Km-oriC", "Input" means the lane where 1.5 µl of a solution containing 0.6 nM each of DCW1-DCW49 and Km-oriC (DCW349) is used. , and "RA" means the lane where a reaction solution containing 0.6 nM each of DCW1-DCW49 and Km-oriC (DCW49) is used.As a result, bound molecules from multiple fragments were detected in samples prepared using DCW1-DCW35 and Km-oriC (DCW35), and in samples prepared using DCW1-DCW49 and Km-oriC (DCW49).

[0208] Then, the reaction mixtures were prepared, after heat treatment and rapid cooling, by adding 0.5 μl of the reaction solution to 4.5 μl of the RCR amplification reaction solution. The reaction mixtures were incubated at 30°C for 16 hours for RCR amplification. Then, 0.5 μl of each product of the RCR amplification reaction was diluted to 4 μl with the reaction buffer, which was obtained by removing only the group of enzymes from the reaction mixture shown in Table 1, and then again incubated at 30°C for 30 minutes. After re-incubation, 2.5 μl of the reaction mixture was subjected to agarose gel electrophoresis and the separated bands were stained with SYBR green.

[0209] The staining results are shown in FIG. 23(b). In this figure, "MK3" means the lane where the DNA ladder marker is used. As a result, as was confirmed in Example 20, in the sample prepared using DCW1-DCW35 and Km-oriC (DCW35), a 36-fragment circular linked molecule was obtained and its amplification product was detected. On the other hand, in samples prepared using DCW1-DCW49 and Km-oriC (DCW49), a thin band was detected at a position where a 50-fragment ring bound molecule band is expected.

[0210] Then, the DNA contained in the reaction solution obtained by the RCR amplification reaction after the coupling reaction was isolated using the reaction solution containing DCW1 - DCW49 and Km-oriC (DCW49) (amplification product of the bound circular molecule obtained by coupling 50 fragments) and assessed the structure of the DNA base sequence.

[0211] In particular, 9 µl of TE buffer (a solution containing 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA) was added to 1 µl of the solution after the RCR reaction, and 1 µl of the resulting diluted solution was mixed with 50 µl of the solution containing competent E. coli cells (E. coli HST08 Premium Electro-Cell, from Takara Bio Inc.). The resulting mixture was subjected to electroporation and transformation. Twelve colonies of the obtained transformants were cultured overnight in 20 ml of liquid LB medium containing 50 μg/ml of kanamycin, and plasmid DNA remaining in Escherichia coli cells cultured in each culture solution was extracted. The DNA concentration in the resulting DNA extract was calculated by measuring the optical density at a wavelength of 260 nm. After calculating the DNA concentration, 15 ng of the extracted DNA was subjected to agarose gel electrophoresis and the separated bands were stained with SYBR green.

[0212] As a result, in 3 (#6, 8, 10) out of 12 colonies, a 50 fragment bound molecule amplification product band (double-stranded cDNA without gaps or nicks) was detected. Then, approximately 15 ng of DNA extracted from these 3 colonies and the colony (No. 12) in which the amplification product band of the bound molecule of 50 fragments could not be detected was subjected to agarose gel electrophoresis using a gel consisting of 1 wt. % agarose, and the separated bands were stained with SYBR green. The staining results are shown in Fig. 24. In this figure, "MK3" means the lane where the DNA ladder marker is used, and "RCR" means the lane where the solution after the RCR reaction is used. "Genome" means a band of E. coli genomic DNA, and "*" means a band of 50-fragment related molecule. As shown in FIG. 24, in the transformants contained in colonies 6, 8, and 10, only bands of Escherichia coli genomic DNA amplification products and bound molecules of 50 fragments were detected.

[0213] Next, the structures of the target DNA sequence were evaluated, which are presumed to be linked circular molecules obtained by linking 50 fragments isolated from colony transformants No. 6, 8 and 10. Based on the nucleotide sequence of the linked molecule of 50 fragments, it was found that digestion with restriction enzyme PciI yielded only four fragments consisting of 10849 bp, 8121 bp, 4771 bp. and 3694 bp, and digestion with the restriction enzyme NcoI gave only six fragments, consisting of 11308 bp, 7741 bp, 4407 bp, 2599 bp, 1123 bp. and 257 bp. Thus, the target DNA, which is assumed to be a circular bound molecule isolated from each transformant, was digested with PciI or NcoI enzymes, and their band patterns were evaluated.

[0214] Specifically, 0.5 μl of 0.03 ng/μl of the extracted DNA was added to 4.5 μl of the RCR amplification reaction solution, and a reaction solution was obtained. The reaction solution was incubated at 30° C. for 16 hours to carry out the RCR amplification reaction. Then, 5 μl of each RCR amplification reaction product was diluted with 20 μl of RCR reaction buffer (in Table 1, this buffer is referred to as "reaction buffer"), followed by incubation again at 30° C. for 30 minutes. After re-incubation, 25 µl of the reaction mixture was added to 25 µl of a solution containing 50 mM Tris-HCl (pH 8.0), 50 mM EDTA, 0.2 wt.% sodium dodecyl sulfate, 100 µg/ml pronase K, 10 wt. % glycerol and 0.2 wt.% bromphenol blue, and then incubated at 37°C for 30 minutes to decompose the RCR reaction proteins. After incubation, an equal amount of PCI solution (TE-saturated phenol:chloroform:isoamyl alcohol = 25:24:1) was added to the solution, and the mixture was vigorously shaken using a vortex mixer, and then centrifuged at 12,000 rpm for 1 minute . The separated aqueous layer was dialyzed against TE buffer using MF (trademark) membrane filters (filter type: 0.05 μm VMWP, manufactured by Merck). The DNA concentration in the DNA solution after dialysis was calculated from the optical density of the DNA solution at a wavelength of 260 nm. After dialysis, 4.5 µl of a solution containing 40 ng of DNA, 1 × NEB 3 buffer, 0.1 wt% BSA was prepared, and then 0.5 µl of 10 U/µl of restriction enzyme PciI (manufactured by Takara Bio Inc.) was added to the solution. .), 10 U/µl NcoI restriction enzyme (manufactured by New England Biolabs), or water. The resulting solution was incubated at 37°C for 30 minutes. After incubation, 2.5 μl of the reaction solution was subjected to agarose gel electrophoresis, and the separated bands were stained with SYBR green.

[0215] The staining results are shown in FIG. 25. In this figure, "MK3" and "MK2" denote the lane where the DNA ladder marker is used, respectively. "PCR product" means the lane where the solution after the RCR reaction is used. "6", "8" and "10" denote lanes where RCR amplification reaction products of DNA extracted from colony transformants 6, 8 and 10, respectively, are used. "-" means the lane where the sample is used without enzyme treatment. As a result, it was confirmed that the cDNA contained in transformants No. 6, 8, 10 are circular linked molecules in which 50 fragments of interest were linked, as indicated by the band patterns of the PciI and NcoI hydrolysates.

[0216] [Example 22]

Linear double-stranded DNA fragments of two or more types containing a DNA fragment with a homologous region located at or near the overhanging 3' end were linked using a RecA family recombinase protein and a 3'→5' exonuclease. In this reaction, the effect of a combination of 3'→5'-exonuclease specific for linear double-stranded DNA and 3'→5'-exonuclease specific for single-stranded DNA was investigated.

[0217] As linked linear double-stranded DNA fragments, a linear double-stranded DNA fragment with 3' overhangs, which was obtained by hydrolysis of pUC4KSceI (PUC4KSceI fragment), was used, and the linear double-stranded DNA fragment was designed to bind in the form of a circular linked molecule with this linear double-stranded DNA fragment (Km-oriC PI-SceI). pUC4KSceI is a plasmid obtained by linking and circularizing a 4 kb fragment and a 500 bp PI-SceI fragment (SEQ ID NO:65) to RA. A 4 kb fragment was obtained by PCR amplification using the PUC4K plasmid as a template and a pair of primers (CTATGCGGCATCAGAGCAG (SEQ ID NO: 63) and GTTAAGCCAGCCCCGACAC (SEQ ID NO: 64)). The Km-oriC PI-SceI is a PCR fragment amplified using the Km-oriC (DCW35) fragment as template and a pair of primers ((tgcgtaagcggggcacatttcattacctctttctccgcacGCTCTGCCAGTGTTACAACC (SEQ ID NO: 66) and taatgtatactatacgaagttattatctatgtcgggtgc (SEQ ID NO: 66) and taatgtatactatacgaagttattatctatgtcgggtgc) IDTGGGGATTATGTAACGGATSE 6 ).

[0218] E. coli F203W RecA mutant was used as the RecA family recombinase protein. Exonuclease III and exonuclease I were used as 3'→5'-exonuclease specific for linear double-stranded DNA and 3'→5'-exonuclease specific for single-stranded DNA, respectively.

[0219] In particular, 1.28 nM of each fragment of PUC4KSceI and Km-oriC PI-SceI, 1.5 μM wild-type RecA, 80 mU/μl exonuclease III, 20 mM Tris-HCl (pH 8.0), 4 mM DTT, 1 mM magnesium acetate, 50 mM potassium glutamate, 100 μM ATP, 150 mM TMAS, 5 wt% PEG8000, 10 v/v DMSO, 4 mM creatine phosphate, 20 ng/μl creatine kinase and 0 U/μl (no addition), 0.3 U/µl, 0.6 U/µl, or 1 U/µl exonuclease I. These reaction solutions were then incubated at 42° C. for 60 minutes to carry out the binding reaction, and then incubated at 65°C for 5 minutes for heat treatment, and then quickly cooled on ice. After heat treatment and rapid cooling, 1.5 μl of the reaction solutions were subjected to agarose gel electrophoresis, and the separated bands were stained with SYBR green.

[0220] The staining results are shown in FIG. 26. In this figure, "Inlet" means the lane where 2 μl of a solution containing 1.28 nM of each PUC4KSceI and Km-oriC PI-SceI fragment is used. In this figure, "pUC4KSceI" means the band of the pUC4KSceI fragment, "Km-oriC" means the Km-oriC band of PI-SceI, and "assembly" means the band of the linked molecule obtained by linking the pUC4KSceI fragment and Km-oriC PI-SceI. As a result, the sample to which more exonuclease I was added had more bound molecules consisting of fragments. It was confirmed that binding efficiency was increased using exonuclease I, which hydrolyzes 3' overhangs, in contrast to exonuclease III, which produces 5' overhangs, which can be easily targeted using exonuclease III.

[0221] [Example 23]

Linear double stranded DNA fragments of two or more types were linked using a RecA family recombinase protein and a 3'→5' exonuclease. In this reaction, the effect of a combination of a 3'→5'-exonuclease specific for linear double-stranded DNA and two types of 3'→5'-exonucleases specific for single-stranded DNA was investigated.

[0222] DCW34 - DCW43 (SEQ ID NO: 34 - SEQ ID NO: 43) were used as linear double-stranded linked DNA fragments. Wild-type E. coli RecA was used as the RecA family recombinase protein. Exonuclease III was used as a 3'→5'-exonuclease specific for linear double-stranded DNA, and exonuclease I and exonuclease T were used as 3'→5'-exonuclease specific for single-stranded DNA.

[0223] In particular, reaction solutions were first prepared consisting of 1 nM each of DCW34-DCW43, 1 μM wild-type RecA, 80 mU/μl exonuclease III, 1 U/μl exonuclease I, 20 mM Trisa-HCl (pH 8 0), 4 mM DTT, 1 mM magnesium acetate, 50 mM potassium glutamate, 100 μM ATP, 150 mM TMAS, 5% w/w PEG8000, 10% v/v DMSO, 20 ng/μl creatine kinase, 4 mM creatine phosphate, and 0 U/µl (no addition), 0.05 U/µl, 0.15 U/µl, or 0.5 U/µl exonuclease T. These reaction solutions were then incubated at 42° C. for 30 minutes to carry out binding reaction, and then incubated at 65°C for 2 minutes for heat treatment, and then quickly cooled on ice. After heat treatment and rapid cooling, 1.5 μl of the reaction solutions were subjected to agarose gel electrophoresis, and the separated bands were stained with SYBR green.

[0224] The staining results are shown in FIG. 27. As a result, in all samples subjected to the binding reaction, a band of a bound molecule was observed, obtained by binding fragments of all 10 types. The number of bound molecules from 2-9 fragments decreased depending on the content of exonuclease T. Based on these results, it was found that the addition of exonuclease I and exonuclease T stimulated the binding reaction of multiple binding fragments.

[0225] [Example 24]

Linear double stranded DNA fragments of two or more types were linked using a RecA family recombinase protein and a 3'→5' exonuclease. The RecA bacteriophage, which is a homologue of the T4 UvsX phage, was used as the RecA family recombinase protein. DCW34 - DCW43 (SEQ ID NO: 34 - SEQ ID NO: 43) were used as linear double-stranded linked DNA fragments.

[0226] Specifically, reaction solutions were first prepared consisting of 1 nM each of DCW34-DCW43, 8 mU/µl, 30 mU/µl, or 80 mU/µl exonuclease III, 1 U/µl exonuclease I, 20 mM Trisa- HCl (pH 8.0), 4 mM DTT, 1 mM magnesium acetate, 50 mM potassium glutamate, 100 μM ATP, 150 mM TMAS, 5% w/w PEG8000, 10% v/v DMSO, 20 ng/μl creatine kinase, 4 mM creatine phosphate and 0 U/µl (no addition), 1 µM or 3 µM UvsX or 1 µM wild-type RecA (control). Then, these reaction solutions were incubated at 42°C for 30 minutes to carry out the coupling reaction, and then incubated at 65°C for 2 minutes for heat treatment, after which they were rapidly cooled on ice. After heat treatment and rapid cooling, 1.5 μl of the reaction solutions were subjected to agarose gel electrophoresis, and the separated bands were stained with SYBR green.

[0227] The staining results are shown in FIG. 28. In this figure, "Inlet" means the lane where 2 µl of a solution containing 1 nM of each of DCW34-DCW43 is used. As a result, a band of a bound molecule obtained by binding fragments of all 10 types in samples obtained after the binding reaction in the presence of 1 μM or 3 μM UvsX and 80 mU/μl of exonuclease III was detected, where these samples were similar to the samples obtained after the binding reaction in in the presence of 1 μM wild-type RecA and 80 mU/μl of exonuclease III. These results confirmed that the binding reaction can be carried out with high binding efficiency even when using UvsX, which is a RecA bacteriophage homologue, unlike wild-type RecA.

[0228] [Example 25]

In the binding reaction of linear double-stranded DNA fragments of two or more types using UvsX and 3'→5'-exonuclease, the effect of using T4 phage together with UvsY was evaluated. DCW34 - DCW43 (SEQ ID NO: 34 - SEQ ID NO: 43) were used as linear double-stranded linked DNA fragments.

[0229] Specifically, reaction solutions were first prepared consisting of 1 nM each of DCW34-DCW43, 3 μM UvsX, 60 mU/μl exonuclease III, 1 U/μl exonuclease I, 20 mM Trisa-HCl (pH 8.0 ), 4 mM DTT, 1 mM magnesium acetate, 50 mM potassium glutamate, 100 μM ATP, 150 mM TMAS, 5% w/w PEG8000, 10% v/v DMSO, 20 ng/μl creatine kinase, 4 mM creatine phosphate, and 0 U. /µl (no addition), 0.1 µM, 0.3 µM or 1 µM UvsY. Then, these reaction solutions were incubated at 42°C for 30 minutes to carry out the coupling reaction, and then incubated at 65°C for 2 minutes for heat treatment, after which they were quickly cooled on ice. After heat treatment and rapid cooling, 1.5 μl of the reaction solutions were subjected to agarose gel electrophoresis, and the separated bands were stained with SYBR green.

[0230] The staining results are shown in FIG. 29. As a result, in all samples subjected to the binding reaction, a band of a bound molecule was detected, obtained by binding fragments of all 10 types. Depending on the content of UvsY, the number of bound molecules obtained by binding fragments of all 10 types increased, while the number of bound molecules from 2-9 fragments decreased. These results showed that using UvsX and UvsY taken together, it is possible to stimulate the binding reaction using exonuclease III and the UvsX stimulator.

Explanation of symbols

[0231] 1a, 1b ... linear double-stranded DNA fragment, H ... homologous region, 2 ... 3'→5' exonuclease, 3 ... RecA family recombinase protein.

[Sequence List]

--->

SEQUENCE LIST

<110> Japan Science and Technology Agency

<120> PC-25879

<130> DNA PRODUCTION METHOD AND DNA FRAGMENT BINDING KIT

<160> 67

<210> 1

<211> 591

<212> DNA

<213> Artificial sequence

<220>

<223> DCW1

<400> 1

ttctaaaacc gtgactgcgg atatcccgat tgtgggggat gctcgccagg tcctcgaaca 60

aatgcttgaa ctcttgtcgc aagaatccgc ccatcaacca ctggatgaga tccgcgactg 120

gtggcagcaa attgaacagt ggcgcgctcg tcagtgcctg aaatatgaca ctcacagtga 180

aaagattaaa ccgcaggcgg tgatcgagac tctttggcgg ttgacgaagg gagacgctta 240

cgtgacgtcc gatgtcgggc agcaccagat gtttgctgca ctttattatc cattcgacaa 300

accgcgtcgc tggatcaatt ccggtggcct cggcacgatg ggttttggtt tacctgcggc 360

actgggcgtc aaaatggcgt tgccagaaga aaccgtggtt tgcgtcactg gcgacggcag 420

tattcagatg aacatccagg aactgtctac cgcgttgcaa tacgagttgc ccgtactggt 480

ggtgaatctc aataaccgct atctggggat ggtgaagcag tggcaggaca tgatctattc 540

cggccgtcat tcacaatctt atatgcaatc gctacccgat ttcgtccgtc t 591

<210> 2

<211> 591

<212> DNA

<213> Artificial sequence

<220>

<223> DCW2

<400> 2

gatctattcc ggccgtcatt cacaatctta tatgcaatcg ctacccgatt tcgtccgtct 60

ggcggaagcc tatgggcatg tcgggatcca gatttctcat ccgcatgagc tggaaagcaa 120

acttagcgag gcgctggaac aggtgcgcaa taatcgcctg gtgtttgttg atgttaccgt 180

cgatggcagc gagcacgtct acccgatgca gattcgcggg ggcggaatgg atgaaatgtg 240

gttaagcaaa acggagagaa cctgattatg cgccggatat tatcagtctt actcgaaaat 300

gaatcaggcg cgttatcccg cgtgattggc ctttttttccc agcgtggcta caacattgaa 360

agcctgaccg ttgcgccaac cgacgatccg acattatcgc gtatgaccat ccagaccgtg 420

ggcgatgaaa aagtacttga gcagatcgaa aagcaattac acaaactggt cgatgtcttg 480

cgcgtgagtg agttggggca gggcgcgcat gttgagcggg aaatcatgct ggtgaaaatt 540

c 591

<210> 3

<211> 591

<212> DNA

<213> Artificial sequence

<220>

<223> DCW3

<400> 3

gtgaaaattc aggccagcgg ttacgggcgt gacgaagtga aacgtaatac ggaaatattc 60

cgtgggcaaa ttatcgatgt cacaccctcg ctttataccg ttcaattagc aggcaccagc 120

ggtaagcttg atgcattttt agcatcgatt cgcgatgtgg cgaaaattgt ggaggttgct 180

cgctctggtg tggtcggact ttcgcgcggc gataaaataa tgcgttgaga atgatctcaa 240

tgcgcaattt acagcccaac atgtcacgtt gggctttttt tgcgaaatca gtgggaacct 300

ggaataaaag cagttgccgc agttaatttt ctgcgcttag atgttaatga atttaaccca 360

taccagtaca atggctatgg tttttacatt ttacgcaagg ggcaattgtg aaactggatg 420

aaatcgctcg gctggcggga gtgtcgcgga ccactgcaag ctatgttatt aacggcaaag 480

cgaagcaata ccgtgtgagc gacaaaaccg ttgaaaaagt catggctgtg gtgcgtgagc 540

acaattacca cccgaacgcc gtggcagctg ggcttcgtgc tggacgcaca c 591

<210> 4

<211> 591

<212> DNA

<213> Artificial sequence

<220>

<223> DCW4

<400> 4

tgcgtgagca caattaccac ccgaacgccg tggcagctgg gcttcgtgct ggacgcacac 60

gttctattgg tcttgtgatc cccgatctgg agaacaccag ctatacccgc atcgctaact 120

atcttgaacg ccaggcgcgg caacggggtt atcaactgct gattgcctgc tcagaagatc 180

agccagacaa cgaaatgcgg tgcattgagc accttttaca gcgtcaggtt gatgccatta 240

ttgtttcgac gtcgttgcct cctgagcatc ctttttatca acgctgggct aacgacccgt 300

tcccgattgt cgcgctggac cgcgccctcg atcgtgaaca cttcaccagc gtggttggtg 360

ccgatcagga tgatgccgaa atgctggcgg aagagttacg taagtttccc gccgagacgg 420

tgctttatct tggtgcgcta ccggagcttt ctgtcagctt cctgcgtgaa caaggtttcc 480

gtactgcctg gaaagatgat ccgcgcgaag tgcatttcct gtatgccaac agctatgagc 540

gggaggcggc tgcccagtta ttcgaaaaat ggctggaaac gcatccgatg c 591

<210> 5

<211> 591

<212> DNA

<213> Artificial sequence

<220>

<223> DCW5

<400> 5

gctatgagcg ggaggcggct gcccagttat tcgaaaaatg gctggaaacg catccgatgc 60

cgcaggcgct gttcacaacg tcgtttgcgt tgttgcaagg agtgatggat gtcacgctgc 120

gtcgcgacgg caaactgcct tctgacctgg caattgccac ctttggcgat aacgaactgc 180

tcgacttctt acagtgtccg gtgctggcag tggctcaacg tcaccgcgat gtcgcagagc 240

gtgtgctgga gattgtcctg gcaagcctgg acgaaccgcg taagccaaaa cctggtttaa 300

cgcgcattaa acgtaatctc tatcgccgcg gcgtgctcag ccgtagctaa gccgcgaaca 360

aaaatacgcg ccaggtgaat ttccctctgg cgcgtagagt acgggactgg acatcaatat 420

gcttaaagta aataagacta ttcctgacta ttattgataa atgcttttaa acccgcccgt 480

taattaactc accagctgaa attcacaata attaagtgat atcgacagcg cgtttttgca 540

ttatttttgtt acatgcggcg atgaattgcc gatttaacaa acacttttct t 591

<210> 6

<211> 591

<212> DNA

<213> Artificial sequence

<220>

<223> DCW6

<400> 6

gtttttgcat tattttgtta catgcggcga tgaattgccg atttaacaaa cacttttctt 60

tgcttttgcg caaacccgct ggcatcaagc gccacacaga cgtaacaagg actgttaacc 120

ggggaagata tgtcctaaaa tgccgctcgc gtcgcaaact gacactttat atttgctgtg 180

gaaaatagtg agtcatttta aaacggtgat gacgatgagg gattttttct tacagctatt 240

cataacgtta atttgcttcg cacgttggac gtaaaataaa caacgctgat attagccgta 300

aacatcgggt tttttacctc ggtatgcctt gtgactggct tgacaagctt ttcctcagct 360

ccgtaaactc ctttcagtgg gaaattgtgg ggcaaagtgg gaataagggg tgaggctggc 420

atgttccgggg gagcaacgtt agtcaatctc gacagcaaag ggcgcttatc agtgcctacc 480

cgttatcggg aacagctgct tgagaacgct gccggtcaaa tggtttgcac cattgacatt 540

tatcacccgt gcctgctgct ttaccccctg cctgaatggg aaattatcga g 591

<210> 7

<211> 591

<212> DNA

<213> Artificial sequence

<220>

<223> DCW7

<400> 7

attgacattt atcacccgtg cctgctgctt taccccctgc ctgaatggga aattatcgag 60

caaaaattat cgcgtctgtc gagcatgaac ccggttgagc gccgtgtgca gcgcctactg 120

ttaggtcatg ccagcgaatg tcagatggat ggcgcaggtc gattgttaat cgcgccagta 180

ctgcggcaac atgccggggct gacaaaagaa gtgatgctgg ttggacagtt caacaagttt 240

gagctgtggg atgaaacaac ctggcatcaa caggtcaagg aagatatcga cgcagagcag 300

ttggctaccg gagacttatc ggagcgactg caggacttgt ctctataaaa tgatggaaaa 360

ctataaacat actacggtgc tgctggatga agccgttaat ggcctcaata tccgtcctga 420

tggcatctac attgatggga cttttggtcg cggtggtcac tcacgtctga tcctctcgca 480

gcttggcgaa gaggggcgtt tgctggcgat cgatcgcgac ccgcaggcta tcgccgttgc 540

gaagactatt gatgatccgc gcttctccat catccacgga cctttctccg c 591

<210> 8

<211> 591

<212> DNA

<213> Artificial sequence

<220>

<223> DCW8

<400> 8

cgccgttgcg aagactattg atgatccgcg cttctccatc atccacggac ctttctccgc 60

gctgggcgaa tacgttgccg agcgcgatct tatcggcaag atcgacggca ttctcctcga 120

tcttggcgtc tcttcaccgc aacttgatga tgctgaacgt ggcttttcct ttatgcgcga 180

tggtccgctg gacatgcgta tggacccaac ccgtgggcag tcagccgctg aatggctaca 240

aaccgcagaa gaagccgata tcgcctgggt attgaaaacc tatggtgaag agcgttttgc 300

360

agaactggcg gaagtcgtgg ctgctgcaac gccggtgaaa gataagttta aacatcccgc 420

gacccgtacc ttccaggcgg tgcgcatttg ggtaaacagt gaactggagg agatagagca 480

ggcgctaaaa agctcgctca acgtgctggc cccgggtggg cggctttcga tcatcagctt 540

g 591

<210> 9

<211> 591

<212> DNA

<213> Artificial sequence

<220>

<223> DCW9

<400> 9

catcagcttc cactcgctgg aagaccgtat tgtgaaacgt tttatgcgtg aaaacagccg 60

cggtccgcaa gttccggcag ggttaccgat gactgaagag cagctcaaaa aactgggtgg 120

ccgtcagctg cgagcactag gcaagttaat gccgggcgaa gaagaggtgg ctgagaaccc 180

tcgtgcccgt agttcagttc tgcgtattgc agagaggacg aatgcatgat cagcagagtg 240

acagaagctc taagcaaagt taaaggatcg atgggaagcc acgagcgcca tgcattgcct 300

ggtgttatcg gtgacgatct tttgcgattt gggaagctgc cactctgcct gttcatttgc 360

attattttga cggcggtgac tgtggtaacc acggcgcacc atacccgttt actgaccgct 420

cagcgcgaac aactggtgct ggagcgagat gctttagaca ttgaatggcg caacctgatc 480

cttgaagaga atgcgctcgg cgaccatagc cgggtggaaa ggatcgccac ggaaaagctg 540

caaatgcagc atgttgatcc gtcacaagaa aatatcgtag tgcaaaaata a 591

<210> 10

<211> 591

<212> DNA

<213> Artificial sequence

<220>

<223> DCW10

<400> 10

gaaaagctgc aaatgcagca tgttgatccg tcacaagaaa atatcgtagt gcaaaaataa 60

ggataaacgc gacgcatgaa agcagcggcg aaaacgcaga aaccaaaacg tcaggaagaa 120

catgccaact ttatcagttg gcgttttgcg ttgttatgcg gctgtattct cctggcgctg 180

gcttttctgc tcggacgcgt agcgtggtta caagttatct ccccggatat gctggtgaaa 240

gagggcgaca tgcgttctct tcgcgttcag caagtttcca cctcccgcgg catgattact 300

gaccgttctg gtcgcccgtt agcggtgagc gtgccggtaa aagcgatttg ggctgacccg 360

aaagaagtgc atgacgctgg cggtatcagc gtcggtgacc gctggaaggc gctggctaac 420

gcgctcaata ttccgctgga tcagctttca gcccgcatta acgccaaccc gaaagggcgc 480

tttatttatc tggcgcgtca ggtgaaccct gacatggcgg actacatcaa aaaactgaaa 540

ctgccgggga ttcatctgcg tgaagagtct cgccgttact atccgtccgg c 591

<210> 11

<211> 591

<212> DNA

<213> Artificial sequence

<220>

<223> DCW11

<400> 11

aaactgaaac tgccggggat tcatctgcgt gaagagtctc gccgttacta tccgtccggc 60

gaagtgactg ctcacctcat cggctttact aacgtcgata gtcaagggat tgagggcgtt 120

gagaagagtt tcgataaatg gcttaccggg cagccggggtg agcgcattgt gcgtaaagac 180

cgctatggtc gcgtaattga agatatttct tctactgaca gccaggcagc gcacaacctg 240

gcgctgagta ttgatgaacg cctgcaggcg ctggtttatc gcgaactgaa caacgcggtg 300

gcctttaaca aggctgaatc tggtagcgcc gtgctggtgg atgtcaacac cggtgaagtg 360

ctggcgatgg ctaacagccc gtcatacaac cctaacaatc tgagcggcac gccgaaagag 420

gcgatgcgta accgtaccat caccgacgtg tttgaaccgg gctcaacggt taaaccgatg 480

gtggtaatga ccgcgttgca acgtggcgtg gtgcgggaaa actcggtact caataccatt 540

ccttatcgaa ttaacggcca cgaaatcaaa gacgtggcac gctacagcga a 591

<210> 12

<211> 591

<212> DNA

<213> Artificial sequence

<220>

<223> DCW12

<400> 12

aataccattc cttatcgaat taacggccac gaaatcaaag acgtggcacg ctacagcgaa 60

ttaaccctga ccggggtatt acagaagtcg agtaacgtcg gtgtttccaa gctggcgtta 120

gcgatgccgt cctcagcgtt agtagatact tactcacgtt ttggactggg aaaagcgacc 180

aatttggggt tggtcggaga acgcagtggc ttatatcctc aaaaacaacg gtggtctgac 240

atagaggg ccaccttctc tttcggctac gggctaatgg taacaccatt acagttagcg 300

360 cgagtctacg caactatcgg cagctacggc atttatcgcc

gaccccccgg ttcccggtga acgtgtcttc ccggaatcca ttgtccgcac tgtggtgcat 420

atgatggaaa gcgtggcgct accaggcggc ggcggcgtga aggcggcgat taaaggctat 480

cgtatcgcca ttaaaaccgg taccgcgaaa aaggtcgggc cggacggtcg ctacatcaat 540

aaatatattg cttataccgc aggcgttgcg cctgcgagtc agccgcgctt c 591

<210> 13

<211> 571

<212> DNA

<213> Artificial sequence

<220>

<223> DCW13b

<400> 13

taaatatatt gcttataccg caggcgttgc gcctgcgagt cagccgcgct tcgcgctggt 60

tgttgttatc aacgatccgc aggcgggtaa atactacggc ggcgccgttt ccgcgccggt 120

ctttggtgcc atcatgggcg gcgtattgcg taccatgaac atcgagccgg atgcgctgac 180

aacgggcgat aaaaatgaat ttgtgattaa tcaaggcgag gggacaggtg gcagatcgta 240

atttgcgcga ccttcttgct ccgtgggtgc cagacgcacc ttcgcgagca ctgcgagaga 300

tgacactcga cagccgtgtg gctgcggcgg gcgatctctt tgtagctgta gtaggtcatc 360

aggcggacgg gcgtcgatat atcccgcagg cgatagcgca aggtgtggct gccattattg 420

cagaggcgaa agatgaggcg accgatggtg aaatccgtga aatgcacggc gtaccggtca 480

tctatctcag ccagctcaac gagcgtttat ctgcactggc gggccgcttt taccatgaac 540

cctctgacaa tttacgtctc gtgggcgtaa c 571

<210> 14

<211> 591

<212> DNA

<213> Artificial sequence

<220>

<223> DCW14

<400> 14

ccgcttttac catgaaccct ctgacaattt acgtctcgtg ggcgtaacgg gcaccaacgg 60

caaaaccacg actacccagc tgttggcgca gtggagccaa ctgcttggcg aaatcagcgc 120

ggtaatgggc accgttggta acggcctgct ggggaaagtg atcccgacag aaaatacaac 180

cggttcggca gtcgatgttc agcatgagct ggcggggctg gtggatcagg gcgcgacgtt 240

ttgcgcaatg gaagtttcct cccacgggct ggtacagcac cgtgtggcgg cattgaaatt 300

tgcggcgtcg gtctttacca acttaagccg cgatcacctt gattatcatg gtgatatgga 360

acactacgaa gccgcgaaat ggctgcttta ttctgagcat cattgcggtc aggcgattat 420

taacgccgac gatgaagtgg gccgccgctg gctggcaaaa ctgccggacg cggttgcggt 480

atcaatggaa gatcatatta atccgaactg tcacggacgc tggttgaaag cgaccgaagt 540

gaactatcac gacagcggtg cgacgattcg ctttagctca agttggggcg a 591

<210> 15

<211> 591

<212> DNA

<213> Artificial sequence

<220>

<223> DCW15

<400> 15

gaccgaagtg aactatcacg acagcggtgc gacgattcgc tttagctcaa gttggggcga 60

tggcgaaatt gaaagccatc tgatgggcgc ttttaacgtc agcaacctgc tgctcgcgct 120

ggcgacactg ttggcactcg gctatccact ggctgatctg ctgaaaaccg ccgcgcgtct 180

gcaaccggtt tgcggacgta tggaagtgtt cactgcgcca ggcaaaccga cggtggtggt 240

ggattacgcg catacgccgg atgcactgga aaaagcctta caggcggcgc gtctgcactg 300

tgcgggcaag ctgtggtgtg tctttggctg tggtggcgat cgcgataaag gtaagcgtcc 360

actgatgggc gcaattgccg aagagtttgc tgacgtggcg gtggtgacgg acgataaccc 420

gcgtaccgaa gaaccgcgtg ccatcatcaa cgatattctg gcgggaatgt tagatgccgg 480

acatgccaaa gtgatggaag gccgtgctga agcggtgact tgcgccgtta tgcaggctaa 540

agagaatgat gtggtactgg tcgcgggcaa aggccatgaa gattaccaga t 591

<210> 16

<211> 591

<212> DNA

<213> Artificial sequence

<220>

<223> DCW16

<400> 16

gcaggctaaa gagaatgatg tggtactggt cgcgggcaaa ggccatgaag attaccagat 60

tgttggcaat cagcgtctgg actactccga tcgcgtcacg gtggcgcgtc tgctgggggt 120

gattgcatga ttagcgtaac ccttagccaa cttaccgaca ttctcaacgg tgaactgcaa 180

ggtgcagata tcacccttga tgctgtaacc actgataccc gaaaactgac gccggggctgc 240

ctgtttgttg ccctgaaagg cgaacgtttt gatgcccacg attttgccga ccaggcgaaa 300

gctggcggcg caggcgcact actggttagc cgtccgctgg acatcgacct gccgcagtta 360

atcgtcaagg atacgcgtct ggcgtttggt gaactggctg catgggttcg ccagcaagtt 420

ccggcgcgcg tggttgctct gacggggtcc tccggcaaaa cctccgttaa agagatgacg 480

gcggcgattt taagccagtg cggcaacacg ctttatacgg caggcaatct caacaacgac 540

atcggtgtac cgatgacgct gttgcgctta acgccggaat acgattacgc a 591

<210> 17

<211> 591

<212> DNA

<213> Artificial sequence

<220>

<223> DCW17

<400> 17

aacaacgaca tcggtgtacc gatgacgctg ttgcgcttaa cgccggaata cgattacgca 60

gttattgaac ttggcgcgaa ccatcaggggc gaaaatagcct ggactgtgag tctgactcgc 120

ccggaagctg cgctggtcaa caacctggca gcggcgcatc tggaaggttt tggctcgctt 180

gcgggtgtcg cgaaagcgaa aggtgaaatc tttagcggcc tgccggaaaa cggtatcgcc 240

attatgaacg ccgacaacaa cgactggctg aactggcaga gcgtaattgg ctcacgcaaa 300

gtgtggcgtt tctcacccaa tgccgccaac agcgatttca ccgccaccaa tatccatgtg 360

acctcgcacg gtacggaatt taccctacaa accccaaccg gtagcgtcga tgttctgctg 420

ccgttgccgg ggcgtcacaa tattgcgaat gcgctggcag ccgctgcgct ctccatgtcc 480

gtgggcgcaa cgcttgatgc tatcaaagcg gggctggcaa atctgaaagc tgttccaggc 540

cgtctgttcc ccatccaact ggcagaaaac cagttgctgc tcgacgactc c 591

<210> 18

<211> 591

<212> DNA

<213> Artificial sequence

<220>

<223> DCW18

<400> 18

gttccaggcc gtctgttccc catccaactg gcagaaaacc agttgctgct cgacgactcc 60

tacaacgcca atgtcggttc aatgactgca gcagtccagg tactggctga aatgccggggc 120

taccgcgtgc tggtggtggg cgatatggcg gaactgggcg ctgaaagcga agcctgccat 180

gtacaggtgg gcgaggcggc aaaagctgct ggtattgacc gcgtgttaag cgtgggtaaa 240

caaagccatg ctatcagcac cgccagcggc gttggcgaac attttgctga taaaactgcg 300

ttaattacgc gtcttaaatt actgattgct gagcaacagg taattacgat tttagttaag 360

ggttcacgta gtgccgccat ggaagaggta gtacgcgctt tacaggagaa tgggacatgt 420

tagtttggct ggccgaacat ttggtcaaat attattccgg ctttaacgtc ttttcctatc 480

tgacgtttcg cgccatcgtc agcctgctga ccgcgctgtt catctcattg tggatgggcc 540

cgcgtatgat tgctcatttg caaaaacttt cctttggtca ggtggtgcgt a 591

<210> 19

<211> 591

<212> DNA

<213> Artificial sequence

<220>

<223> DCW19

<400> 19

ggatgggccc gcgtatgatt gctcatttgc aaaaactttc ctttggtcag gtggtgcgta 60

acgacggtcc tgaatcacac ttcagcaagc gcggtacgcc gaccatgggc gggattatga 120

tcctgacggc gattgtgatc tccgtactgc tgtgggctta cccgtccaat ccgtacgtct 180

ggtgcgtgtt ggtggtgctg gtaggttacg gtgttattgg ctttgttgat gattatcgca 240

aagtggtgcg taaagacacc aaagggttga tcgctcgttg gaagtatttc tggatgtcgg 300

tcattgcgct gggtgtcgcc ttcgccctgt accttgccgg caaagacacg cccgcaacgc 360

agctggtggt cccattcttt aaagatgtga tgccgcagct ggggctgttc tacattctgc 420

tggcttactt cgtcattgtg ggtactggca acgcggtaaa cctgaccgat ggtctcgacg 480

gcctggcaat tatgccgacc gtatttgtcg ccggtggttt tgcgctggtg gcgtgggcga 540

ccggcaatat gaactttgcc agctacttgc atataccgta tctgcgacac g 591

<210> 20

<211> 591

<212> DNA

<213> Artificial sequence

<220>

<223> DCW20

<400> 20

cgtgggcgac cggcaatatg aactttgcca gctacttgca tataccgtat ctgcgacacg 60

ccggggaact ggttattgtc tgtaccgcga tagtcggggc aggactgggc ttcctgtggt 120

ttaacaccta tccggcgcag gtctttatgg gcgatgtagg ttcgctggcg ttaggtggtg 180

cgttaggcat tatcgccgta ctgctacgtc aggaattcct gctggtgatt atggggggcg 240

tgttcgtggt agaaacgctt tctgtcatcc tgcaggtcgg ctcctttaaa ctgcgcggac 300

aacgtatttt ccgcatggca ccgattcatc accactatga actgaaaggc tggccggaac 360

cgcgcgtcat tgtgcgtttc tggattattt cgctgatgct ggttctgatt ggtctggcaa 420

cgctgaaggt acgttaatca tggctgatta tcagggtaaa aatgtcgtca ttatcggcct 480

gggcctcacc gggctttcct gcgtggactt ttttcctcgct cgcggtgtga cgccgcgcgt 540

tatggatacg cgtatgacac cgcctggcct ggataaatta cccgaagccg t 591

<210> 21

<211> 591

<212> DNA

<213> Artificial sequence

<220>

<223> DCW21

<400> 21

gccgcgcgtt atggatacgc gtatgacacc gcctggcctg gataaattac ccgaagccgt 60

agaacgccac acgggcagtc tgaatgatga atggctgatg gcggcagatc tgattgtcgc 120

cagtcccggt attgcactgg cgcatccatc cttaagcgct gccgctgatg cgggaatcga 180

aatcgttggc gatatcgagc tgttctgtcg cgaagcacaa gcaccgattg tggcgattac 240

cggttctaac ggcaaaagca cggtcaccac gctagtgggt gaaatggcga aagcggcggg 300

ggttaacgtt ggtgtgggtg gcaatattgg cctgcctgcg ttgatgctac tggatgatga 360

gtgtgaactg tacgtgctgg aactgtcgag cttccagctg gaaaccacct ccagcttaca 420

ggcggtagca gcgaccattc tgaacgtgac tgaagatcat atggatcgct atccgtttgg 480

540

taatgctgat gatgccttaa caatgccgat tcgcggtgcg gatgaacgct g 591

<210> 22

<211> 591

<212> DNA

<213> Artificial sequence

<220>

<223> DCW22

<400> 22

ttgcgtggtt aatgctgatg atgccttaac aatgccgatt cgcggtgcgg atgaacgctg 60

cgtcagcttt ggcgtcaaca tgggtgacta tcacctgaat catcagcagg gcgaaacctg 120

gctgcgggtt aaaggcgaga aagtgctgaa tgtgaaagag atgaaacttt cggggcagca 180

taactacacc aatgcgctgg cggcgctggc gctggcagat gctgcagggt taccgcgtgc 240

cagcagcctg aaagcgttaa ccacattcac tggtctgccg catcgctttg aagttgtgct 300

ggagcataac ggcgtacgtt ggattaacga ttcgaaagcg accaacgtcg gcagtacgga 360

agcggcgctg aatggcctgc acgtagacgg cacactgcat ttgttgctgg gtggcgatgg 420

taaatcggcg gactttagcc cactggcgcg ttacctgaat ggcgataacg tacgtctgta 480

ttgtttcggt cgtgacggcg cgcagctggc ggcgctacgc cgggaagtgg cagaacaaac 540

cgaaactatg gaacaggcga tgcgcttgct ggctccgcgt gttcagccgg g 591

<210> 23

<211> 591

<212> DNA

<213> Artificial sequence

<220>

<223> DCW23

<400> 23

agaacaaacc gaaactatgg aacaggcgat gcgcttgctg gctccgcgtg ttcagccggg 60

cgatatggtt ctgctctccc cagcctgtgc cagccttgat cagttcaaga actttgaaca 120

acgaggcaat gagtttgccc gtctggcgaa ggagttaggt tgatgcgttt atctctccct 180

cgcctgaaaa tgccgcgcct gccaggattc agtatcctgg tctggatctc cacggcgcta 240

aagggctggg tgatgggctc gcgggaaaaa gtaccgaca gcctgatcat gtacgatcgc 300

accttactgt ggctgacctt cggcctcgcg gcgattggct ttatcatggt gacctcggcg 360

tcaatgccca tagggcaacg cttaaccaac gatccgttct tcttcgcgaa gcgtgatggt 420

gtctatctga ttttggcgtt tattctggcg atcattacgc tgcgtctgcc gatggagttc 480

tggcaacgct acagtgccac gatgctgctc ggatctatca tcctgctgat gatcgtcctg 540

gtagtgggta gctcggttaa aggggcatcg cgttggatcg atctcggttt g 591

<210> 24

<211> 589

<212> DNA

<213> Artificial sequence

<220>

<223> DCW24b

<400> 24

atcgtcctgg tagtgggtag ctcggttaaa ggggcatcgc gttggatcga tctcggtttg 60

ctgcgtatcc agcctgcgga gctgacaaaa ctgtcgctgt tttgctatat cgccaactat 120

ctggtgcgta aaggcgacga agtacgtaat aacctgcgcg gcttcctgaa accgatgggc 180

gtgattctgg tgttggcagt gttactgctg gcacagccag accttggtac ggtggtggtg 240

ttgtttgtga ctacgctggc gatgttgttc ctggcgggag cgaaattgtg gcagttcatt 300

gccattatcg gtatgggcat ttcagcggtt gtgttgctga tactcgccga accgtaccgt 360

atccgccgtg ttaccgcatt ctggaacccg tgggaagatc cctttggcag cggctatcag 420

ttaacgcaat cgctgatggc gtttggtcgc ggcgaacttt gggggcaagg tttaggtaac 480

tcggtacaaa aactggagta tctgccggaa gcgcacactg actttatttt cgccattatc 540

ggcgaagaac tggggtatgt cggtgtggtg ctggcacttt taatggtat 589

<210> 25

<211> 591

<212> DNA

<213> Artificial sequence

<220>

<223> DCW25

<400> 25

gccattatcg gcgaagaact ggggtatgtc ggtgtggtgc tggcactttt aatggtattc 60

ttcgtcgctt ttcgcgcgat gtcgattggc cgtaaagcat tagaaattga ccaccgtttt 120

tccggttttc tcgcctgttc tattggcatc tggtttagct tccaggcgct ggttaacgta 180

ggcgcggcgg cggggatgtt accgaccaaa ggtctgacat tgccgctgat cagttacggt 240

ggttcgagct tactgattat gtcgacagcc atcatgatgc tgttgcgtat tgattatgaa 300

acgcgtctgg agaaagcgca ggcgtttgta cgaggttcac gatgagtggt caaggaaagc 360

gattaatggt gatggcaggc ggaaccggtg gacatgtatt cccgggactg gcggttgcgc 420

accatctaat ggctcagggt tggcaagttc gctggctggg gactgccgac cgtatggaag 480

cggacttagt gccaaaacat ggcatcgaaa ttgatttcat tcgtatctct ggtctgcgtg 540

gaaaaggtat aaaagcactg atagctgccc cgctgcgtat cttcaacgcc t 591

<210> 26

<211> 591

<212> DNA

<213> Artificial sequence

<220>

<223> DCW26

<400> 26

gtctgcgtgg aaaaggtata aaagcactga tagctgcccc gctgcgtatc ttcaacgcct 60

ggcgtcaggc gcgggcgatt atgaaagcgt acaaacctga cgtggtgctc ggtatgggag 120

gctacgtgtc aggtccaggt ggtctggccg cgtggtcgtt aggcattccg gttgtacttc 180

atgaacaaaa cggtattgcg ggcttaacca ataaatggct ggcgaagatt gccaccaaag 240

tgatgcaggc gtttccaggt gctttcccta atgcggaagt agtgggtaac ccggtgcgta 300

ccgatgtgtt ggcgctgccg ttgccgcagc aacgtttggc tggacgtgaa ggtccggttc 360

gtgtgctggt agtgggtggt tctcaggggcg cacgcattct taaccagaca atgccgcagg 420

ttgctgcgaa actgggtgat tcagtcacta tctggcatca gagcggcaaa ggttcgcaac 480

aatccgttga acaggcgtat gccgaagcgg ggcaaccgca gcataaagtg acggaattta 540

ttgatgatat ggcggcggcg tatgcgtggg cggatgtcgt cgtttgccgc t 591

<210> 27

<211> 591

<212> DNA

<213> Artificial sequence

<220>

<223> DCW27

<400> 27

60

ccggtgcgtt aacggtgagt gaaatcgccg cggcaggact accggcgttg tttgtgccgt 120

ttcaacataa agaccgccag caatactgga atgcgctacc gctggaaaaa gcgggcgcag 180

ccaaaattat cgagcagcca cagcttagcg tggatgctgt cgccaacacc ctggccgggt 240

ggtcgcgaga aaccttatta accatggcag aacgcgcccg cgctgcatcc attccggatg 300

ccaccgagcg agtggcaaat gaagtgagcc gggttgcccg ggcgtaattg tagcgatgcc 360

ttttgcatcg tatgaattta agaagttaat ggcgtaaaga atgaatacac aacaattggc 420

aaaactgcgt tccatcgtgc ccgaaatgcg tcgcgttcgg cacatacatt ttgtcggcat 480

tggtggtgcc ggtatggggcg gtattgccga agttctggcc aatgaaggtt atcagatcag 540

tggttccgat ttagcgccaa atccggtcac gcagcagtta atgaatctgg g 591

<210> 28

<211> 591

<212> DNA

<213> Artificial sequence

<220>

<223> DCW28

<400> 28

tcagatcagt ggttccgatt tagcgccaaa tccggtcacg cagcagttaa tgaatctggg 60

120

ttccagcgcg atttctgccg ataacccgga aattgtcgcc gctcatgaag cgcgtattcc 180

ggtgatccgt cgtgccgaaa tgctggctga gttaatgcgt tttcgtcatg gcatcgccat 240

tgccggaacg cacggcaaaa cgacaaccac cgcgatggtt tccagcatct acgcagaagc 300

ggggctcgac ccaaccttcg ttaacggcgg gctggtaaaa gcggcggggg ttcatgcgcg 360

tttggggcat ggtcggtacc tgattgccga agcagatgag agtgatgcat cgttcctgca 420

tctgcaaccg atggtggcga ttgtcaccaa tatcgaagcc gaccacatgg atacctacca 480

gggcgacttt gagaatttaa aacagacttt tattaattttt ctgcacaacc tgccgtttta 540

cggtcgtgcg gtgatgtgtg ttgatgatcc ggtgatccgc gaattgttac c 591

<210> 29

<211> 591

<212> DNA

<213> Artificial sequence

<220>

<223> DCW29

<400> 29

gccgttttac ggtcgtgcgg tgatgtgtgt tgatgatccg gtgatccgcg aattgttacc 60

gcgagtgggg cgtcagacca cgacttacgg cttcagcgaa gatgccgacg tgcgtgtaga 120

agattatcag cagattggcc cgcaggggca ctttacgctg ctgcgccagg acaaagagcc 180

gatgcgcgtc accctgaatg cgccaggtcg tcataacgcg ctgaacgccg cagctgcggt 240

tgcggttgct acggaagagg gcattgacga cgaggctatt ttgcggggcgc ttgaaagctt 300

ccaggggact ggtcgccgtt ttgatttcct cggtgaattc ccgctggagc cagtgaatgg 360

taaaagcggt acggcaatgc tggtcgatga ctacggccac cacccgacgg aagtggacgc 420

caccattaaa gcggcgcgcg caggctggcc ggataaaaac ctggtaatgc tgtttcagcc 480

gcaccgtttt acccgtacgc gcgacctgta tgatgatttc gccaatgtgc tgacgcaggt 540

tgataccctg ttgatgctgg aagtgtatcc ggctggcgaa gcgccaattc c 591

<210> 30

<211> 591

<212> DNA

<213> Artificial sequence

<220>

<223> DCW30

<400> 30

gacgcaggtt gataccctgt tgatgctgga agtgtatccg gctggcgaag cgccaattcc 60

gggagcggac agccgttcgc tgtgtcgcac aattcgtgga cgtgggaaaa ttgatcccat 120

tctggtgccg gatccggcgc gggtagccga gatgctggca ccggtattaa ccggtaacga 180

cctgattctc gttcaggggg ctggtaatat tggaaaaatt gcccgttctt tagctgaaat 240

caaactgaag ccgcaaactc cggaggaaga acaacatgac tgataaaatc gcggtcctgt 300

tgggtgggac ctccgctgag cgggaagttt ctctgaattc tggcgcagcg gtgttagccg 360

gactgcgtga aggcggtatt gacgcgtatc ctgtcgaccc gaaagaagtc gacgtgacgc 420

aactgaagtc gatgggcttt cagaaagtgt ttatcgcgct acacggtcgc ggcggtgaag 480

atggtacgct gcaggggatg ctcgagctga tgggcttgcc ttataccggga agcggagtga 540

tggcatctgc gctttcaatg gataaactac gcagcaaact tctatggcaa g 591

<210> 31

<211> 591

<212> DNA

<213> Artificial sequence

<220>

<223> DCW31

<400> 31

gcggagtgat ggcatctgcg ctttcaatgg ataaactacg cagcaaactt ctatggcaag 60

gtgccggttt accggtcgcg ccgtgggtag cgttaacccg cgcagagttt gaaaaaggcc 120

tgagcgataa gcagttagca gaaatttctg ctctgggttt gccggttatc gttaagccga 180

gccgcgaagg ttccagtgtg ggaatgtcaa aagtagtagc agaaaatgct ctacaagatg 240

cattaagatt ggcatttcag cacgatgaag aagtattgat tgaaaaatgg ctaagtgggc 300

cggagttcac ggttgcgata ctcggtgaag aaattttacc gtcaatacgt attcaaccgt 360

ccggaacctt ctatgattat gaggcgaagt atctctctga tgagacacag tatttctgcc 420

ccgcaggtct ggaagcgtca caagaggcca atttgcaggc attagtgctg aaagcatgga 480

cgacgttagg ttgcaaagga tggggacgta ttgacgttat gctggacagc gatggacagt 540

tttatctgct ggaagccaat acctcaccgg gtatgaccag ccacagcctg g 591

<210> 32

<211> 579

<212> DNA

<213> Artificial sequence

<220>

<223> DCW32b

<400> 32

atggacagtt ttatctgctg gaagccaata cctcaccgggg tatgaccagc cacagcctgg 60

tgccgatggc ggcacgtcag gcaggtatga gcttctcgca gttggtagta cgaattctgg 120

aactggcgga ctaatatgtc gcaggctgct ctgaacacgc gaaacagcga agaagaggtt 180

tcttctcgcc gcaataatgg aacgcgtctg gcggggatcc ttttcctgct gaccgtttta 240

300

ccgctctcaa agctggtgtt gaccggtgaa cgccattaca cacgtaatga cgatatccgg 360

cagtcgatcc tggcattggg tgagccgggt acctttatga cccaggatgt caacatcatc 420

cagacgcaaa tagaacaacg cctgccgtgg attaagcagg tgagcgtcag aaagcagtgg 480

cctgatgaat tgaagattca tctggttgaa tatgtgccga ttgcgcggtg gaatgatcaa 540

catatggtag acgcggaagg aaataccttc agcgtgccg 579

<210> 33

<211> 591

<212> DNA

<213> Artificial sequence

<220>

<223> DCW33

<400> 33

aatgatcaac atatggtaga cgcggaagga aataccttca gcgtgccgcc agaacgcacc 60

agcaagcagg tgcttccaat gctgtatggc ccggaaggca gcgccaatga agtgttgcag 120

ggctatcgcg aaatggggca gatgctggca aaggacagat ttactctgaa ggaagcggcg 180

atgaccgcgc ggcgttcctg gcagttgacg ctgaataacg atattaagct caatcttggc 240

cggggcgata cgatgaaacg tttggctcgc tttgtagaac tttatccggt tttacagcag 300

caggcgcaaa ccgatggcaa acggattagc tacgttgatt tgcgttatga ctctggagcg 360

gcagtaggct gggcgccctt gccgccagag gaatctactc agcaacaaaa tcaggcacag 420

gcagaacaac aatgatcaag gcgacggaca gaaaactggt agtaggactg gagattggta 480

ccgcgaaggt tgccgcttta gtaggggaag ttctgcccga cggtatggtc aatatcattg 540

gcgtgggcag ctgcccgtcg cgtggtatgg ataaaggcgg ggtgaacgac c 591

<210> 34

<211> 591

<212> DNA

<213> Artificial sequence

<220>

<223> DCW34

<400> 34

atatcattgg cgtgggcagc tgcccgtcgc gtggtatgga taaaggcggg gtgaacgacc 60

tcgaatccgt ggtcaagtgc gtacaacgcg ccattgacca ggcagaattg atggcagatt 120

gtcagatctc ttcggtatat ctggcgcttt ctggtaagca catcagctgc cagaatgaaa 180

ttggtatggt gcctatttct gaagaagaag tgacgcaaga agatgtggaa aacgtcgtcc 240

ataccgcgaa atcggtgcgt gtgcgcgatg agcatcgtgt gctgcatgtg atcccgcaag 300

agtatgcgat tgactatcag gaagggatca agaatccggt aggactttcg ggcgtgcgga 360

tgcaggcaaa agtgcacctg atcacatgtc acaacgatat ggcgaaaaac atcgtcaaag 420

cggttgaacg ttgtgggctg aaagttgacc aactgatatt tgccggactg gcatcaagtt 480

attcggtatt gacggaagat gaacgtgaac tgggtgtctg cgtcgtcgat atcggtggtg 540

gtacaatgga tatcgccgtt tataccggtg gggcattgcg cccactaag g 591

<210> 35

<211> 591

<212> DNA

<213> Artificial sequence

<220>

<223> DCW35

<400> 35

tcggtggtgg tacaatggat atcgccgttt ataccggtgg ggcattgcgc cacactaagg 60

taattcctta tgctggcaat gtcgtgacca gtgatatcgc ttacgccttt ggcacgccgc 120

caagcgacgc cgaagcgatt aaagttcgcc acggttgtgc gctgggttcc atcgttggaa 180

aagatgagag cgtggaagtg ccgagcgtag gtggtcgtcc gccacggagt ctgcaacgtc 240

agacactggc agaggtgatc gagccgcgct ataccgagct gctcaacctg gtcaacgaag 300

agatattgca gttgcaggaa aagcttcgcc aacaaggggt taaacatcac ctggcggcag 360

gcattgtatt aaccggtggc gcagcgcaga tcgaaggtct tgcagcctgt gctcagcgcg 420

tgtttcatac gcaagtgcgt atcggcgcgc cgctgaacat taccggttta acggattatg 480

ctcaggagcc gtattattcg acggcggtgg gattgcttca ctatgggaaa gagtcacatc 540

ttaacggtga agctgaagta gaaaaacgtg ttacagcatc agttggctcg t 591

<210> 36

<211> 591

<212> DNA

<213> Artificial sequence

<220>

<223> DCW36

<400> 36

agtcacatct taacggtgaa gctgaagtag aaaaacgtgt tacagcatca gttggctcgt 60

ggatcaagcg actcaatagt tggctgcgaa aagagtttta atttttatga ggccgacgat 120

gattacggcc tcaggcgaca ggcacaaatc ggagagaaac tatgtttgaa ccaatggaac 180

ttaccaatga cgcggtgatt aaagtcatcg gcgtcggcgg cggcggcggt aatgctgttg 240

aacacatggt gcgcgagcgc attgaaggtg ttgaattctt cgcggtaaat accgatgcac 300

aagcgctgcg taaaacagcg gttggacaga cgattcaaat cggtagcggt atcaccaaag 360

gactgggcgc tggcgctaat ccagaagttg gccgcaatgc ggctgatgag gatcgcgatg 420

cattgcgtgc ggcgctggaa ggtgcagaca tggtctttat tgctgcgggt atgggtggtg 480

gtaccggtac aggtgcagca ccagtcgtcg ctgaagtggc aaaagatttg ggtatcctga 540

ccgttgctgt cgtcactaag cctttcaact ttgaaggcaa gaagcgtatg g 591

<210> 37

<211> 591

<212> DNA

<213> Artificial sequence

<220>

<223> DCW37

<400> 37

gtatcctgac cgttgctgtc gtcactaagc ctttcaactt tgaaggcaag aagcgtatgg 60

cattcgcgga gcaggggatc actgaactgt ccaagcatgt ggactctctg atcactatcc 120

cgaacgacaa actgctgaaa gttctgggcc gcggtatctc cctgctggat gcgtttggcg 180

cagcgaacga tgtactgaaa ggcgctgtgc aaggtatcgc tgaactgatt actcgtccgg 240

gtttgatgaa cgtggacttt gcagacgtac gcaccgtaat gtctgagatg ggctacgcaa 300

tgatgggttc tggcgtggcg agcggtgaag accgtgcgga agaagctgct gaaatggcta 360

tctcttctcc gctgctggaa gatatcgacc tgtctggcgc gcgcggcgtg ctggttaaca 420

tcacggcggg cttcgacctg cgtctggatg agttcgaaac ggtaggtaac accatccgtg 480

catttgcttc cgacaacgcg actgtggtta tcggtacttc tcttgacccg gatatgaatg 540

acgagctgcg cgtaaccgtt gttgcgacag gtatcggcat ggacaaacgt c 591

<210> 38

<211> 591

<212> DNA

<213> Artificial sequence

<220>

<223> DCW38

<400> 38

atatgaatga cgagctgcgc gtaaccgttg ttgcgacagg tatcggcatg gacaaacgtc 60

ctgaaatcac tctggtgacc aataagcagg ttcagcagcc agtgatggat cgctaccagc 120

agcatgggat ggctccgctg acccaggagc agaagccggt tgctaaagtc gtgaatgaca 180

atgcgccgca aactgcgaaa gagccggatt atctggatat cccagcattc ctgcgtaagc 240

aagctgatta agaattgact ggaatttggg tttcgaggct ctttgtgcta aactggcccg 300

ccgaatgtat agtacacttc ggttggatag gtaatttggc gagataatac gatgatcaaa 360

caaaggacac ttaaacgtat cgttcaggcg acgggtgtcg gtttacatac cggcaagaaa 420

gtcaccctga cgttacgccc tgcgccggcc aacaccgggg tcatctatcg tcgcaccgac 480

ttgaatccac cggtagattt cccggccgat gccaaatctg tgcgtgatac catgctctgt 540

acgtgtctgg tcaacgagca tgatgtacgg atttcaaccg tagagcacct c 591

<210> 39

<211> 591

<212> DNA

<213> Artificial sequence

<220>

<223> DCW39

<400> 39

atgctctgta cgtgtctggt caacgagcat gatgtacgga tttcaaccgt agagcacctc 60

aatgctgctc tcgcgggctt gggcatcgat aacattgtta tcgaagttaa cgcgccggaa 120

atcccgatca tggacggcag cgccgctccg tttgtatacc tgctgcttga cgccggtatc 180

gacgagttga actgcgccaa aaaatttgtt cgcatcaaag agactgttcg tgtcgaagat 240

ggcgataagt gggctgaatt taagccgtac aatggttttt cgctggattt caccatcgat 300

tttaaccatc cggctattga ttccagcaac cagcgctatg cgatgaactt ctccgctgat 360

gcgtttatgc gccagatcag ccgtgcgcgt acgttcggtt tcatgcgtga tatcgaatat 420

ctgcagtccc gtggtttgtg cctgggcggc agcttcgatt gtgccatcgt tgttgacgat 480

tatcgcgtac tgaacgaaga cggcctgcgt tttgaagacg aatttgtgcg tcacaaaatg 540

ctcgatgcga tcggtgactt gttcatgtgt ggtcacaata ttattggtgc a 591

<210> 40

<211> 591

<212> DNA

<213> Artificial sequence

<220>

<223> DCW40

<400> 40

cacaaaatgc tcgatgcgat cggtgacttg ttcatgtgtg gtcacaatat tattggtgca 60

tttaccgctt ataaatccgg tcatgcactg aataacaaac tgctgcaggc tgtcctggcg 120

aaacaggaag cctgggaata tgtgaccttc caggacgacg cagaactgcc gttggccttc 180

aaagcgcctt cagctgtact ggcataacga catttatact gtcgtataaa attcgactgg 240

caaatctggc actctctccg gccaggtgaa ccagtcgttt ttttttgaat tttataagag 300

ctataaaaaa cggtgcgaac gctgttttct taagcacttt tccgcacaac ttatcttcat 360

tcgtgctgtg gactgcaggc tttaatgata agatttgtgc gctaaatacg tttgaatatg 420

atcgggatgg caataacgtg agtggaatac tgacgcgctg gcgacagttt ggtaaacgct 480

acttctggcc gcatctctta ttagggatgg ttgcggcgag tttaggtttg cctgcgctca 540

gcaacgccgc cgaaccaaac gcgcccgcaa aagcgacaac ccgcaaccac g 591

<210> 41

<211> 591

<212> DNA

<213> Artificial sequence

<220>

<223> DCW41

<400> 41

ctgcgctcag caacgccgcc gaaccaaacg cgcccgcaaa agcgacaacc cgcaaccacg 60

agccttcagc caaagttaac tttggtcaat tggccttgct ggaagcgaac acacgccgcc 120

cgaattcgaa ctattccgtt gattactggc atcaacatgc cattcgcacg gtaatccgtc 180

atctttcttt cgcaatggca ccgcaaacac tgcccgttgc tgaagaatct ttgcctcttc 240

aggcgcaaca tcttgcatta ctggatacgc tcagcgcgct gctgacccag gaaggcacgc 300

cgtctgaaaa gggttatcgc attgattatg cgcattttac cccacaagca aaattcagca 360

cgcccgtctg gataagccag gcgcaaggca tccgtgctgg ccctcaacgc ctcacctaac 420

aacaataaac ctttacttca ttttattaac tccgcaacgc ggggcgtttg agattttatt 480

atgctaatca aattgttaac taaagttttc ggtagtcgta acgatcgcac cctgcgccgg 540

atgcgcaaag tggtcaacat catcaatgcc atggaaccgg agatggaaaa a 591

<210> 42

<211> 591

<212> DNA

<213> Artificial sequence

<220>

<223> DCW42

<400> 42

ctgcgccgga tgcgcaaagt ggtcaacatc atcaatgcca tggaaccggga gatggaaaaa 60

ctctccgacg aagaactgaa agggaaaacc gcagagtttc gtgcacgtct ggaaaaaggc 120

gaagtgctgg aaaatctgat ccgggaagct ttcgccgtgg tacgtgaggc aagtaagcgc 180

gtctttggta tgcgtcactt cgacgttcag ttactcggcg gtatggttct taacgaacgc 240

tgcatcgccg aaatgcgtac cggtgaagga aaaaccctga ccgcaacgct gcctgcttac 300

ctgaacgcac taaccggtaa aggcgtgcac gtagttaccg tcaacgacta cctggcgcaa 360

cgtgacgccg aaaacaaccg tccgctgttt gaattccttg gcctgactgt cggtatcaac 420

ctgccgggca tgccagcacc ggcaaagcgc gaagcttacg cagctgacat cacttacggt 480

acgaacaacg aatacggctt tgactacctg cgcgacaaca tggcgttcag ccctgaagaa 540

cgtgtacagc gtaaactgca ctatgcgctg gtggacgaag tggactccat c 591

<210> 43

<211> 591

<212> DNA

<213> Artificial sequence

<220>

<223> DCW43

<400> 43

cctgaagaac gtgtacagcg taaactgcac tatgcgctgg tggacgaagt ggactccatc 60

ctgatcgatg aagcgcgtac accgctgatc atttccggcc cggcagaaga cagctcggaa 120

atgtataaac gcgtgaataa aattattccg cacctgatcc gtcaggaaaa agaagactcc 180

gaaaccttcc agggcgaagg ccacttctcg gtggacgaaa aatctcgcca ggtgaacctg 240

accgaacgtg gtctggtgct gattgaagaa ctgctggtga aaggggcat catggatgaa 300

ggggagtctc tgtactctcc ggccaacatc atgctgatgc accacgtaac ggcggcgctg 360

cgcgctcatg cgctgtttac ccgtgacgtc gactacatcg ttaaagatgg tgaagttatc 420

atcgttgacg aacacaccgg tcgtaccatg cagggccgtc gctggtccga tggtctgcac 480

caggctgtgg aagcgaaaga aggtgtgcag atccagaacg aaaaccaaac gctggcttcg 540

atcaccttcc agaactactt ccgtctgtat gaaaaactgg cggggatgac c 591

<210> 44

<211> 591

<212> DNA

<213> Artificial sequence

<220>

<223> DCW44

<400> 44

ctggcttcga tcaccttcca gaactacttc cgtctgtatg aaaaactggc ggggatgacc 60

ggtactgctg ataccgaagc tttcgaattt agctcaatct acaagctgga taccgtcgtt 120

gttccgacca accgtccaat gattcgtaaa gatctgccgg acctggtcta catgactgaa 180

gcggaaaaaa ttcaggcgat cattgaagat atcaaagaac gtactgcgaa aggccagccg 240

gtgctggtgg gtactatctc catcgaaaaa tcggagctgg tgtcaaacga actgaccaaa 300

360

gctcaggcag gttatccggc tgcggtgact atcgcgacca atatggcggg tcgtggtaca 420

gatattgtgc tcggtggtag ctggcaggca gaagttgccg cgctggaaaa tccgaccgca 480

gagcaaattg aaaaaattaa agccgactgg caggtacgtc acgatgcggt actggaagca 540

ggtggcctgc atatcatcgg taccgagcgt cacgaatccc gtcgtatcga t 591

<210> 45

<211> 591

<212> DNA

<213> Artificial sequence

<220>

<223> DCW45

<400> 45

ctggaagcag gtggcctgca tatcatcggt accgagcgtc acgaatcccg tcgtatcgat 60

aaccagttgc gcggtcgttc tggtcgtcag ggggatgctg gttcttcccg tttctacctg 120

tcgatggaag atgcgctgat gcgtattttt gcttccgacc gagtatccgg catgatgcgt 180

aaactgggta tgaagccagg cgaagccatt gaacacccgt gggtgactaa agcgattgcc 240

aacgcccagc gtaaagttga aagccgtaac ttcgacattc gtaagcaact gctggaatat 300

gatgacgtgg ctaacgatca gcgtcgcgcc atttactccc agcgtaacga actgttggat 360

gtcagcgatg tgagcgaaac cattaacagc attcgtgaag atgtgttcaa agcgaccatt 420

gatgcctaca ttccaccaca gtcgctggaa gaaatgtggg atattccggg gctgcaggaa 480

cgtctgaaga acgatttcga cctcgatttg ccaattgccg agtggctgga taaagaacca 540

gaactgcatg aagagacgct gcgtgagcgc attctggcgc agtccatcga a 591

<210> 46

<211> 591

<212> DNA

<213> Artificial sequence

<220>

<223> DCW46

<400> 46

aaagaaccag aactgcatga agagacgctg cgtgagcgca ttctggcgca gtccatcgaa 60

gtgtatcagc gtaaagaaga agtggttggt gctgagatga tgcgtcactt cgagaaaggc 120

gtcatgctgc aaacgcttga ctccctgtgg aaagagcacc tggcagcgat ggactatctg 180

cgtcagggta tccacctgcg tggctacgca cagaaagatc cgaagcagga atacaaacgt 240

gaatcgttct ccatgtttgc agcgatgctg gagtcgttga aatatgaagt tatcagtacg 300

ctgagcaaag ttcaggtacg tatgcctgaa gaggttgagg agctggaaca acagcgtcgt 360

atggaagccg agcgtttagc gcaaatgcag cagcttagcc atcaggatga cgactctgca 420

gccgcagctg cactggcggc gcaaaccggga gagcgcaaag taggacgtaa cgatccttgc 480

ccgtgcggtt ctggtaaaaa atacaagcag tgccatggcc gcctgcaata aaagctaact 540

gttgaagtaa aaggtgcagg attctgcgcc ttttttatag gtttaagaca a 591

<210> 47

<211> 575

<212> DNA

<213> Artificial sequence

<220>

<223> DCW47b

<400> 47

gctaactgtt gaagtaaaag gtgcaggatt ctgcgccttt tttataggtt taagacaatg 60

aaaaagctgc aaattgcggt aggtattatt cgcaacgaga acaatgaaat ctttataacg 120

cgtcgcgcag cagatgcgca catggcgaat aaactggagt ttcccggcgg taaaattgaa 180

atgggtgaaa cgccggaaca ggcggtggtg cgtgaacttc aggaagaagt cgggattacc 240

ccccaacatt tttcgctatt tgaaaaactg gaatatgaat tcccggacag gcatataaca 300

ctgtggtttt ggctggtcga acgctgggaa ggggagccgt ggggtaaaga agggcaaccc 360

ggtgagtgga tgtcgctggt cggtcttaat gccgatgatt ttccgccagc caatgaaccg 420

gtaattgcga agcttaaacg tctgtaggtc agataaggcg ttttcgccgc atccgacatt 480

cgcacacgat gcctgatgcg acgctggcgc gtcttatcag gcctaaaggg atttctaact 540

cattgataaa tttgtttttg taggtcggat aaggc 575

<210> 48

<211> 559

<212> DNA

<213> Artificial sequence

<220>

<223> DCW48b

<400> 48

tttgtttttg taggtcggat aaggcgttca cgccgcatcc gacatttgca caagatgcct 60

gatgcgacgc tgtccgcgtc ttatcaggcc tacgtgcggc atcagacaaa tgtcactgct 120

ttggttcttc gctccagtca tcgctttcgg aaagatcgcc actgctgggg attcgttttt 180

cttcagcagc ccattctccg aggtcgatca gctgacaacg tttggagcaa aatggccgaa 240

acgggctgat ttcaccccac accgtaatag 300

tttctgacat ttttactcct tagcaacagg ccagttcgaa atccagacgt tccggtacct 360

gtccgttttc agtgtccagc ggcataaaac gaatggcaaa acggctctta tgtccggaaa 420

tttgcggata aagctgtgaa tcgagcgaca gattcaggcg cagcaagtcg gcatcgccac 480

cgttatcctg ataaaaacca ttcaggctgg tttgtttacg gaagggggcc gactggcgaa 540

ttaaatccag caccatggt 559

<210> 49

<211> 558

<212> DNA

<213> Artificial sequence

<220>

<223> DCW49

<400> 49

tttgtttacg gaagggggcc gactggcgaa ttaaatccag caccatggta agtgcctggg 60

tgagcgggtt caggctggca atccaggttt ctacctggct gtcgcgctgc gcctggggta 120

gatgcagcca aatgtgcaat gtaggtaaat caaagctgca acagccgcct gggatgctca 180

gtcgctgacg caccagagca atcaaacgat cttcacgcag aaattgcccg atacgcggcg 240

cggaaattaa tacgctcccc gccgctttta actgctgaat taatgcttca atacggctct 300

ggtccacgcc aggcacgcca atccaggtct ggagtttacg ttgctgccgg tcaagttctt 360

tcaacagctc agtgcggact tcgccgcgct cgaaaacatc cagtaattca ctgacattac 420

ggaagaaatg cagcgcgcca gcgtggtcaa cgatgggtaa attaacggtg agttgctgaa 480

tcaaaaactc aatgcgcagc catgtacgca ttttttcatt tagtggatgt tcaaaaagga 540

cctgggtctg cattacgg 558

<210> 50

<211> 1298

<212> DNA

<213> Artificial sequence

<220>

<223> Cm-oriC (DCW20)

<400> 50

gccgcgcgtt atggatacgc gtatgacacc gcctggcctg gataaattac ccgaagccgt 60

gtcggcttga gaaagacctg agtatgttgt aactaaaggt gcgcataatg tatattatgt 120

taaatggatc ctgggtatta aaaagaagat ctatttattt agagatctgt tctattgtga 180

240

cctggaaagg atcattaact gtgaatgatc ggtgatcctg gaccgtataa gctgggatca 300

gaatgagggg ttatacacaa ctcaaaaact gaacaacagt tgttctttgg ataactaccg 360

gttgatccaa gcttcctgac agagttatcc acagtagatc gcacgatctg tataacttcg 420

tatagcatac attatacgaa gttatcttta gttacaacat actccattct tctgccggat 480

cttcttacgc cccgccctgc cactcatcgc agtactgttg taattcatta agcattctgc 540

cgacatggaa gccatcacag acggcatgat gaacctgaat cgccagcggc atcagcacct 600

tgtcgccttg cgtataatat ttgcccatgg tgaaaacggg ggcgaagaag ttgtccatat 660

tggccacgtt taaatcaaaa ctggtgaaac tcacccaggg attggctgag acgaaaaaca 720

tattctcaat aaacccttta gggaaatagg ccaggttttc accgtaacac gccacatctt 780

gcgaatatat gtgtagaaac tgccggaaat cgtcgtggta ttcactccag agcgatgaaa 840

acgtttcagt ttgctcatgg aaaacggtgt aacaagggtg aacactatcc catatcacca 900

gctcaccgtc tttcattgcc atacggaatt ccggatgagc attcatcagg cgggcaagaa 960

tgtgaataaa ggccggataa aacttgtgct tatttttctt tacggtcttt aaaaaggccg 1020

taatatccag ctgaacggtc tggttatagg tacattgagc aactgactga aatgcctcaa 1080

aatgttcttt acgatgccat tgggatatat caacggtggt atatccagtg atttttttct 1140

ccattttagc ttccttagct cctgaaaatc tcgataactc aaaaaatacg cccggtagtg 1200

1260

atcccgattg tgggggatgc tcgccaggtc ctcgaaca 1298

<210> 51

<211> 1298

<212> DNA

<213> Artificial sequence

<220>

<223> Cm-oriC (DCW25)

<400> 51

gtctgcgtgg aaaaggtata aaagcactga tagctgcccc gctgcgtatc ttcaacgcct 60

gtcggcttga gaaagacctg agtatgttgt aactaaaggt gcgcataatg tatattatgt 120

taaatggatc ctgggtatta aaaagaagat ctatttattt agagatctgt tctattgtga 180

240

cctggaaagg atcattaact gtgaatgatc ggtgatcctg gaccgtataa gctgggatca 300

gaatgagggg ttatacacaa ctcaaaaact gaacaacagt tgttctttgg ataactaccg 360

gttgatccaa gcttcctgac agagttatcc acagtagatc gcacgatctg tataacttcg 420

tatagcatac attatacgaa gttatcttta gttacaacat actccattct tctgccggat 480

cttcttacgc cccgccctgc cactcatcgc agtactgttg taattcatta agcattctgc 540

cgacatggaa gccatcacag acggcatgat gaacctgaat cgccagcggc atcagcacct 600

tgtcgccttg cgtataatat ttgcccatgg tgaaaacggg ggcgaagaag ttgtccatat 660

tggccacgtt taaatcaaaa ctggtgaaac tcacccaggg attggctgag acgaaaaaca 720

tattctcaat aaacccttta gggaaatagg ccaggttttc accgtaacac gccacatctt 780

gcgaatatat gtgtagaaac tgccggaaat cgtcgtggta ttcactccag agcgatgaaa 840

acgtttcagt ttgctcatgg aaaacggtgt aacaagggtg aacactatcc catatcacca 900

gctcaccgtc tttcattgcc atacggaatt ccggatgagc attcatcagg cgggcaagaa 960

tgtgaataaa ggccggataa aacttgtgct tatttttctt tacggtcttt aaaaaggccg 1020

taatatccag ctgaacggtc tggttatagg tacattgagc aactgactga aatgcctcaa 1080

aatgttcttt acgatgccat tgggatatat caacggtggt atatccagtg atttttttct 1140

ccattttagc ttccttagct cctgaaaatc tcgataactc aaaaaatacg cccggtagtg 1200

1260

atcccgattg tgggggatgc tcgccaggtc ctcgaaca 1298

<210> 52

<211> 1509

<212> DNA

<213> Artificial sequence

<220>

<223> Km-oriC (DCW25)

<400> 52

gtctgcgtgg aaaaggtata aaagcactga tagctgcccc gctgcgtatc ttcaacgcct 60

gtcggcttga gaaagacctg agtatgttgt aactaaaggt gcgcataatg tatattatgt 120

taaatggatc ctgggtatta aaaagaagat ctatttattt agagatctgt tctattgtga 180

240

cctggaaagg atcattaact gtgaatgatc ggtgatcctg gaccgtataa gctgggatca 300

gaatgagggg ttatacacaa ctcaaaaact gaacaacagt tgttctttgg ataactaccg 360

gttgatccaa gcttcctgac agagttatcc acagtagatc gcacgatctg tataacttcg 420

tatagcatac attatacgaa gttatcttta gttacaacat actccattct tctgccggat 480

cttcgggaaa gccacgttgt gtctcaaaat ctctgatgtt acattgcaca agataaaaat 540

atatcatcat gaacaataaa actgtctgct tacataaaca gtaatacaag gggtgttatg 600

agccatattc aacgggaaac gtcttgctcg aggccgcgat taaattccaa catggatgct 660

gatttatatg ggtataaatg ggctcgcgat aatgtcgggc aatcaggtgc gacaatctat 720

cgattgtatg ggaagcccga tgcgccagag ttgtttctga aacatggcaa aggtagcgtt 780

gccaatgatg ttacagatga gatggtcaga ctaaactggc tgacggaatt tatgcctctt 840

ccgaccatca agcattttat ccgtactcct gatgatgcat ggttactcac cactgcgatc 900

cccgggaaaa cagcattcca ggtattagaa gaatatcctg attcaggtga aaatattgtt 960

gatgcgctgg cagtgttcct gcgccggttg cattcgattc ctgtttgtaa ttgtcctttt 1020

1080

gatgcgagtg attttgatga cgagcgtaat ggctggcctg ttgaacaagt ctggaaagaa 1140

atgcataagc ttttgccatt ctcaccggat tcagtcgtca ctcatggtga tttctcactt 1200

1260

atcgcagacc gatacgga tcttgccatc ctatggaact gcctcggtga gttttctcct 1320

1380

cagtttcatt tgatgctcga tgagtttttc taatcagaat tggttaattg gttgtaacac 1440

tggcagagct tctaaaaccg tgactgcgga tatcccgatt gtgggggatg ctcgccaggt 1500

cctcgaaca 1509

<210> 53

<211> 1509

<212> DNA

<213> Artificial sequence

<220>

<223> Km-oriC (DCW35)

<400> 53

agtcacatct taacggtgaa gctgaagtag aaaaacgtgt tacagcatca gttggctcgt 60

gtcggcttga gaaagacctg agtatgttgt aactaaaggt gcgcataatg tatattatgt 120

taaatggatc ctgggtatta aaaagaagat ctatttattt agagatctgt tctattgtga 180

240

cctggaaagg atcattaact gtgaatgatc ggtgatcctg gaccgtataa gctgggatca 300

gaatgagggg ttatacacaa ctcaaaaact gaacaacagt tgttctttgg ataactaccg 360

gttgatccaa gcttcctgac agagttatcc acagtagatc gcacgatctg tataacttcg 420

tatagcatac attatacgaa gttatcttta gttacaacat actccattct tctgccggat 480

cttcgggaaa gccacgttgt gtctcaaaat ctctgatgtt acattgcaca agataaaaat 540

atatcatcat gaacaataaa actgtctgct tacataaaca gtaatacaag gggtgttatg 600

agccatattc aacgggaaac gtcttgctcg aggccgcgat taaattccaa catggatgct 660

gatttatatg ggtataaatg ggctcgcgat aatgtcgggc aatcaggtgc gacaatctat 720

cgattgtatg ggaagcccga tgcgccagag ttgtttctga aacatggcaa aggtagcgtt 780

gccaatgatg ttacagatga gatggtcaga ctaaactggc tgacggaatt tatgcctctt 840

ccgaccatca agcattttat ccgtactcct gatgatgcat ggttactcac cactgcgatc 900

cccgggaaaa cagcattcca ggtattagaa gaatatcctg attcaggtga aaatattgtt 960

gatgcgctgg cagtgttcct gcgccggttg cattcgattc ctgtttgtaa ttgtcctttt 1020

1080

gatgcgagtg attttgatga cgagcgtaat ggctggcctg ttgaacaagt ctggaaagaa 1140

atgcataagc ttttgccatt ctcaccggat tcagtcgtca ctcatggtga tttctcactt 1200

1260

atcgcagacc gatacgga tcttgccatc ctatggaact gcctcggtga gttttctcct 1320

1380

cagtttcatt tgatgctcga tgagtttttc taatcagaat tggttaattg gttgtaacac 1440

tggcagagct tctaaaaccg tgactgcgga tatcccgatt gtgggggatg ctcgccaggt 1500

cctcgaaca 1509

<210> 54

<211> 3100

<212> DNA

<213> Artificial sequence

<220>

<223> Lter1

<400> 54

aatagaaaac tgccagtgcg caacctgtac cggttcatgc aatttaaaca gacaaatcgg 60

tctgccattg atcatatttt ctgacaaaag ctcgccacac tgttcaaacc cgcgacgcca 120

gcgttcagca gtggcgtttt gatggcaacg caaagaaatg tgatcggcag tcagcggagt 180

gatattcaac cccagacggc gggaaagttc atctaatgcg tggataaatc gcggtaaatc 240

cgatgcaata tcctgcagct cgtcgataga ttgccagttc gccataatca ctcttcgtct 300

ttcagtaaaa gcgttaattt accctgttgc cctgtgccaa ccaaccgctg atttcacgcc 360

gcttctgatg caatagtgaa aacggcaata cgccacgcgc acgttgctga cgaaaacagc 420

catttgcagt atactcccgc cctaatttct ttaactggtg cgggcaattt ttgctcgctt 480

catcaatgta aggtattccg gtgaatattc aggctcttct ctcagaaaaa gtccgtcagg 540

ccatgattgc ggcaggcgcg cctgcggatt gcgaaccgca ggttcgtcag tcagcaaaag 600

ttcagttcgg cgactatcag gctaacggca tgatggcagt tgctaaaaaa ctgggtatgg 660

caccgcgaca attagcagag caggtgctga ctcatctgga tcttaacggt atcgccagca 720

aagttgagat cgccggtcca ggctttatca acattttcct tgatccggca ttcctggctg 780

aacatgttca gcaggcgctg gcgtccgatc gtctcggtgt tgctacgcca gaaaaacaga 840

ccattgtggt tgactactct gcgccaaacg tggcgaaaga gatgcatgtc ggtcacctgc 900

gctctaccat tattggtgac gcagcagtgc gtactctgga gttcctcggt cacaaagtga 960

ttcgcgcaaa ccacgtcggc gactggggca ctcagttcgg tatgctgatt gcatggctgg 1020

aaaagcagca gcaggaaaac gccggtgaaa tggagctggc tgaccttgaa ggtttctacc 1080

gcgatgcgaa aaagcattac gatgaagatg aagagttcgc cgagcgcgca cgtaactacg 1140

tggtaaaact gcaaagcggt gacgaatatt tccgcgagat gtggcgcaaa ctggtcgaca 1200

tcaccatgac gcagaaccag atcacctacg atcgtctcaa cgtgacgctg acccgtgatg 1260

acgtgatggg cgaaagcctc tacaacccga tgctgccagg aattgtggcg gatctcaaag 1320

ccaaaggtct ggcagtagaa agcgaagggg cgaccgtcgt attccttgat gagtttaaaa 1380

acaaggaagg cgaaccgatg ggcgtgatca ttcagaagaa agatggcggc tatctctaca 1440

ccaccactga tatcgcctgt gcgaaatatc gttatgaaac actgcatgcc gatcgcgtgc 1500

tgtattacat cgactcccgt cagcatcaac acctgatgca ggcatgggcg atcgtccgta 1560

aagcaggcta tgtaccggaa tccgtaccgc tggaacacca catgttcggc atgatgctgg 1620

1680 gtaaagacgg caaaccgttc aaaacccgcg cgggtggtac

tggatgaagc cctggaacgt gcacgccgtc tggtggcaga aaagaacccg gatatgccag 1740

ccgacgagct ggaaaaactg gctaacgcgg ttggtattgg tgcggtgaaa tatgcggatc 1800

tctccaaaaa ccgcaccacg gactacatct tcgactggga caacatgctg gcgtttgagg 1860

gtaataccgc gccatacatg cagtatgcat acacgcgtgt attgtccgtg ttccgtaaag 1920

cagaaattga cgaagagcaa ctggctgcag ctccggttat catccgtgaa gatcgtgaag 1980

cgcaactggc agctcgcctg ctgcagtttg aagaaaccct caccgtggtt gcccgtgaag 2040

gcacgccgca tgtaatgtgt gcttacctgt acgatctggc cggtctgttc tctggcttct 2100

2160

tggcacaact gacggcgaag acgctgaagc tgggtctgga tacgctgggt attgagactg 2220

tagagcgtat gtaatcgatt tttcgtgaga gtgaagcctg atcatgacct ggcaaagcag 2280

aaagcacaat cctcaggcgg gttaccgggt caaaggtggt cgtccggcgc tggtggtggt 2340

gtttctctgt ggtattgctg tgattggcgt gcaatttttg attgcggcag ggttgttacc 2400

agaagtgggg tgatcagata gcctcaaatt ccttattggg tgccagaatt aacgctgaca 2460

cccaatttgg cctcttaatg caggcagcac tgcttaaatt tcttaccact accgcacgggg 2520

caaggatcgt tacgccccgg tttctcttct gctttgatcg gttgctgaac agctttttcc 2580

tgcggatgcg ccatccagta cgcatgtaga tcaagcgccg ccagtcgaat ggcatctacg 2640

ctctcttcaa acgcttctgg cgacatcttt tctacccgct cgaagttttc ctcagtaccg 2700

tgcagcgcaa tcgcctccag cgctggtttt aacgaatcgg gcaacgttga ccagtcagaa 2760

agtgccacgc cccgcatata gccaaagcac cactcctcaa caatcgtcag ctcgctgcca 2820

tcaacttctc gcaagccgaa taacggctca aactgctccg ggaattcgtt cagacgctct 2880

gcggtatcgg ccatatgttg aaaagccaga ttcataaagc gcgtcatctc tttctctgac 2940

gcccagcgcg gcacatagtc agccccaccc cacacggcaa ccagccactg ttccggttca 3000

atctcttgcg gagaactcaa caccgccgtc aataaaccgt ccagctccgc cacatcaagg 3060

atggcgtggt cagtgttgta tttggtcaga atatcgtcca 3100

<210> 55

<211> 3100

<212> DNA

<213> Artificial sequence

<220>

<223> Lter2

<400> 55

ccagctccgc cacatcaagg atggcgtggt cagtgttgta tttggtcaga atatcgtcca 60

gccattccaa ctcactttcg tttaacggtc ccgttttcat acgcttttcc ttgtggatct 120

caactcgcca gcacctatct tacatgccgg tccgtatcag agatactttt tgagtggctt 180

tgctggtgat taaaaattaa ggagggtgta acgacaagtt gcaggcacaa aaaaaccacc 240

300

cccggagcgg gacttgaacc cgcacagcgc gaacgccgag ggattttaaa tcccttgtgt 360

ctaccgattc caccatccgg gctcgggaag aaagtggagg cgcgttccgg agtcgaaccg 420

gactagacgg atttgcaatc cgctacataa ccgctttgtt aacgcgccaa attcttcagg 480

cctttcagcc agacatccgc ttgacgccga tgtcttttaa actggagcgg gaaacgagac 540

tcgaactcgc gaccccgacc ttggcaaggt cgtgctctac caactgagct attcccgcat 600

660

cgatgcgttg cattctactt acctggcgcg atgagtcaac gatatttttc accacttttg 720

atcgtttgct gaaaattacg ccgaaacgat cactgatcaa gcaaatctgc acgcgcagcg 780

ctcaaatatt gcaacattga ccacagagtc agtaccgcag ccacaaagaa aagtgcaata 840

ccggcgtact caacccaaat gttcggacgc cacagcagcc atgccaacgc caccatctgg 900

gcagtggttt tcactttccc aatccaggag acggccacgc tactgcgttt acccaactcc 960

gccatccatt cgcgtagcgc agaaataata atttcacggg cgatcatcgt tgccgccggt 1020

aatgtcaccc accagctgtg ataatgctcg gttaccagca ccatggcgat agccacgaga 1080

actttatctg ccacagggtc aaggaaagca ccaaaccggg tactctggtt ccagcggcgt 1140

gccagaaaac catcgaacca gtcagtcacc gccgcgacgc agaaaatgag cgcggcggca 1200

aacggcgacc aggtgacagg cagataaaag accaatacaa agaatgggat aaggatgaca 1260

cggaacagtg taagcaacgt agggatatta aattgcataa tgacgggtaa ctatctgttg 1320

1380

tttctgccag accttgcgaa atacccggca cttttaatcg tgggctgttt gcttccttgg 1440

gcggatacga gttttattat cgtcttaatg atttccacat attaaaagca agtatgcttt 1500

1560

1620

1680

attccagtgg gacgtaaatg ggccaatatt gttgctaaaa aaacggctaa agacggtgca 1740

acgtctaaaa tttatgcaaa attcggtgta gaaatctatg ctgctgctaa acaaggtgaa 1800

cccgatccag aattaaacac atctttaaaa ttcgttattg aacgtgcaaa gcaggcacaa 1860

gttccaaagc acgttattga taaagcaatt gataaagcca aaggcggcgg agatgaaacg 1920

ttcgtgcagg gacgttatga aggctttggt cctaatggct caatgattat cgccgagaca 1980

ttgacttcaa atgttaaccg tacgattgct aacgttcgca caattttcaa taaaaaaggc 2040

ggcaatatcg gagcggcagg ttctgtcagc tatatgtttg acaatacggg tgtgattgta 2100

2160

cgtgatgtga ctgaagaaga aggtaacatt gttatttata ctgaacctac tgaccttcat 2220

aaaggaatcg cggctctaaa agcagctgga atcactgagt tctcaacaac agaattagaa 2280

atgattgctc aatctgaagt tgagctttcc ccagaagatt tagaaatctt tgaagggctt 2340

gttgatgccc ttgaagatga cgacgatgta caaaaagttt atcataacgt cgcaaatctc 2400

taattatctt ttaaagaaat ctgtctttac ggcagatttc tttaatctca tataattctt 2460

ataaaaaata taatattcaa ctcgtcatat tgattatacc cccccgttcc cagagaaata 2520

atatttatta aaattccagt tcttcttttt ctgattacag aaggcaaagt ggcaattacg 2580

catagtttcc cgataaagac gcgatagcga catcccgcat aaggcatttt tctctttatc 2640

tttgtacggt acttcatgga acagagtttt tgaccttgcg aatcgtgatg tctgttgggg 2700

agggacaatt tgctcactga agcgtgagac tcgattaagc gcacgaaaca cagaaatcaa 2760

2820

tttgagactc atagagatgt ctcaaaacta aaatttggct cctctgactg gactcgaacc 2880

agtgacatac ggattaacag tccgccgttc taccgactga actacagagg aatcgtgaga 2940

acgaggcgaa tattagcgat gcccacccac aatgtcaaag cctgtttttt aaatttgaaa 3000

3060

acttttgcgg ctcgggtcga gggtatttcc atagccaacg 3100

<210> 56

<211> 3100

<212> DNA

<213> Artificial sequence

<220>

<223> Lter3

<400> 56

cacaactggc tttttttcac acttttgcgg ctcgggtcga gggtatttcc atagccaacg 60

tccagtaacc attcgccagt aaaacagcac ggcccgcaca gcccagtcgg caaacattcc 120

catccagaca ccaaccacac cccagccaag catgattccc agcacataac cgactacaac 180

ccgacaaccc cacatgctca acatcgaaac ccacatggcg taacgggcat cacgagcacc 240

tttaaatcca gcgggtagca cccatgaggc ggaccaaata ggcataaata aagcatttag 300

ccaaatcaga atcacaacga catgtttaac ctgtggatcc tgggtgtaaa acgatgccat 360

aaccccggca aagggagccg ttagccaggc gatggccgtt aatccaagag tggaaagcca 420

gaacacatgc cgcaactgaa tctctgcttg cgctatctgc cctaccccca accttcggcc 480

tgtaatgatc gtagaagcag agccgagcgc acttccgggt aagttgataa gagccgcaat 540

tgaaaacgcg ataaaatttc cggcaataac actggtcccc atcccggcaa cgaacatttg 600

ggttaataac cgaccactgg taaataacac tgattcgaca ctcgcgggaa taccaatccc 660

catgacttcc cagataatgc taaaattcag cggtttaaaa tagctcttta acgaaatcct 720

tagcgcagga ttaaaaccaa tcgccagcac ccacaaaatt gcaactgcgc caatataacg 780

agaaatggtt aaacccagcc ctgccccgac aaatcccagt cccggccagg agaaaaggcc 840

gtaaatcaat atgccgctaa taataatatt aagaatattc aggctaccgt taatcaatag 900

cggtattttc gtattccctg caccacgaag tgccccgcta ccaataagag tgatggcagc 960

tgctggataa ctgagtaccg tcagctccag ataagtcaac gccagtgctt taacttctgt 1020

cgtggcatca cccgcgacga aatcaataat ttgttcgcca aaatgatgaa taagcgttgc 1080

1140

tcgtcgatcc cgcttaccga gactaaatgc cacaacgaca gtagtaccaa gatcgatagc 1200

agcaaaaaaa gccataatga ccatattgaa gctgtccgcc aatcccacgc cggccatcgc 1260

atcttttccc agccagctga ccagaaaagt gctcagaacc cccatcaaca ggacacaggc 1320

attctccatg aagataggaa cagcaagcgg ggttatctcg cgccagaaca acactttgta 1380

gctcttgcgt ttagcgtgcc agcgagtgcc gtgaacaacc tggcgtaaag cagaggagat 1440

attcaaagcc gaccttaatt gcagaaagtg aaaccacatt tcaaataatg agggagaatc 1500

agcaaagctg caaagatttt cgccaacaaa ttgtctgcaa atgcaacaaa ctgttgatag 1560

aaacggcaaa cagttgggga atttaaaaat cgggtttgac aaaagatttt tcgccgttaa 1620

gatgtgcctc aacaacgatt cctctgtagt tcagtcggta gaacggcggga ctgttaatcc 1680

gtatgtcact ggttcgagtc cagtcagagg agccaaattc ctgaaaagcc cgcttttata 1740

gcgggatttt tgctatatct gataatcaat ttcctcttca ctgctttcca tcacctgccg 1800

cttgatatcc tcaactgaca gtcctgcatt acaaagttcc agaaagcgcc agacatagtt 1860

acgctgaagt tgtcctcgct tcagtcccaa ccagacagta ttagcatcaa aaagatgccg 1920

cgtatccagg cggattaaat tctcttcctc ttgttcgcca ctggattgct cggcaactaa 1980

tccgatccca agcccaagag caacataggt tttaatgaca tcagaatcct gcgcacttaa 2040

tacaatatct gccagcaaac ctttgcgggc aaatgcgtca tcaatacgtg agcgccccgt 2100

aatcccctgt cggtaagtga ttaacggcca cttcgctatt gattccagcg tcaatggtga 2160

aatttgcgtc aagggatgat cgtgtggaac aagcaaacta tggtgccaac gaaaccacgg 2220

gaaggcgacg agctgcgggt cattactcaa acgctcgctg gcgataccaa tatcagcttc 2280

gccattttgc aacaatgtcg caatttcctg tggcgtcccc tggattagct cgagccgaac 2340

ctccgggaaa agttcgcgaa aagctttaat gacctctggc aagctataac gtgcctgagt 2400

atgcgtcgtt gcaatagtga gaacgccaga cgtatcgttg gtaaacaggt ctgcaagccg 2460

acgaacatta ctggcttcat tcagaatacg ttctgcaatg accagtaatg ctttgcccgg 2520

ttcagtcatg cccagcagtc gcttacctcg tcgaacaaat atttcgatgc caagttcatc 2580

ctccagttcc cgaatatgac ggctgacgcc tgactgtgag gtaaaaagca tattcgcaac 2640

ctctgtcagg ttgtaatcct gacgtgcagc ctcgcggatt atctttagtt gttggaaatt 2700

cacggtaaac tccggggcagt tcagatttcc cgttattgtt aaagtctaat gcccggcata 2760

acaaataata aaaacccgca tcttattcca tcccgatata acacttagct caccaattgc 2820

cactgccttt tttccatcac tggagaacta atcactgaca ttaacaactc tttcactgcc 2880

tgtgcctgtg gcgataagtt cgctctggcg ggtaaattta atgacaaaga gagactcatg 2940

gaaggagtgg taatgcgtga catccaccca tttactgcgc cacataacga acgcgcggcc 3000

gattcgggta atactgcaac gcccatgccg ctggcaatcg ctgcggtaag cgtggcaata 3060

gactcaattt caccaataac ttttgccgtg agtcgccgta 3100

<210> 57

<211> 3100

<212> DNA

<213> Artificial sequence

<220>

<223> Lter4

<400> 57

ctgcggtaag cgtggcaata gactcaattt caccaataac ttttgccgtg agtcgccgta 60

gggaaaaagc ctcatcaaca cgaagtctaa tagcactgta atcactgggg agaaagaggt 120

tcatttgcgc aatagcattc acatcaacgc tttgccccgg gcaatcttga gttcctacca 180

gaaaaagatc ttctttcagc aaagcctgac tggatacacc agccacaggg gaatgctcat 240

aaatcaccgc catatcgagt tggtgattta tcaatttttc gttaagcact gcaccactat 300

tttcatgaag atagataacg atctccggaa attcagcgcg aaccgcctgt aataagggca 360

tggtgatgga tgacgcagcg gttcctggtg caaagccaat cgagacttgc cccgataatg 420

cctgaccaac gttatgcacc gccagttggg cctgttcaca ctgacgtaaa atggcccgcg 480

catgggtata gagaattttt ccggcgtctg ttggtgtaac gccccgcttt gtacggatca 540

aaagttgttg atttaactca ccttccagtg tggcaacctg ctggctgagc gctggttgtg 600

660

atttcaggcg tctgaagttc atgttgcctc cggtttttaa gaatcggccc aagtgccgcc 720

attacttaca accagattgc aagatgcttg ccagttttat tttggtgttg atgtacaagc 780

taaccaactg tcaaataaga gattatgata gattcgtcat ttgctccttt aatcagctgt 840

cgcgttcccc tgccctataa aggagggta tgcaccacga tggttcatta cccaataaga 900

ttgaaagctc accactttgt tgaaattgac agcaaacaaa caaaaaaatg catttcaccc 960

tttgacatca cctgcactg ccattaatat gcgccccgtt cacacgattc ctctgtagtt 1020

1080

gccaaattca aaaaagcctg ctttctagca ggctttttgc tttctaatta ccaacgctct 1140

taaaacatct gtcttgaacc agaactaatt tgcacaggca ttcccgatcg acgttgcaac 1200

1260

gttaccggca atggcacttt tcggtcagac tcatattctg cacgattgcg cgacaatggc 1320

tcatgaactt ccagccagtt cgagccatct ggttcagtgg tgtattttac tggctggtcg 1380

ataatttgca cacgcgtccc aacaggaaca ttatcaaaca gatatttgat atcgtcattg 1440

cgcagacgaa tacagccctg acttacccgg agcccaatac caaaattggc attggtacca 1500

tggatggcat acaacctgcc aatataaatc gcgtacagcc ccatgggatt atcggggccc 1560

gcaggaacaa atgcgggcaa actctcccct cgtttcgcat attcgcgccg agtgttcggc 1620

gttggcgtcc aggttggagc ttcttgttta cgttcaacgg tagtcaccca gttacgcggg 1680

gtttctcgcc cagcctggcc gataccaata ggaaagactt ccacagtatt actgtctggt 1740

gggtagtaat aaagacgcat ctcagcgacg ttaacaacaa tccctttacg aacagtgtcg 1800

ggcaaaatca gttgctgcgg aatggtgagt tgcgagccag acttcggcaa aaaaacatca 1860

gcgcccgggt tcgcttccag catgttactt aacccttgcc cgtattgtgc ggcaaaagtc 1920

tccagcggct gggtattgtg atcaggaaca gttacagtaa acgactgccc cactaaacgg 1980

ctaccctctg gaggtaatgg ataagttacc gccaggctag tatggctggc aaaaagcaga 2040

gcaaatgagc aaagaatatt tacacgacgc atcatgtccc tttcctatgt cgcgaaagct 2100

atccgttaag tatagctttt atcagacttt tcgtttttaa ctgttcaaat cagaagtcgt 2160

attccccggt agaacattgt tcgttcatcc cgactccttt tttgtataga taaaccatca 2220

gctgatagtt tacctgaaga atatagagaa gtacttactt aacattttcc catttggtac 2280

tatctaaccc cttttcacta ttaagaagta atgcctacta tgactcaagt cgcgaagaaa 2340

attctggtga cgtgcgcact gccgtacgct aacggctcaa tccacctcgg ccatatgctg 2400

gagcacatcc aggctgatgt ctgggtccgt taccagcgaa tgcgcggcca cgaggtcaac 2460

ttcatctgcg ccgacgatgc ccacggtaca ccgatcatgc tgaaagctca gcagcttggt 2520

atcaccccgg agcagatgat tggcgaaatg agtcaggagc atcagactga tttcgcaggc 2580

tttaacatca gctatgacaa ctatcactcg acgcacagcg aagagaaccg ccagttgtca 2640

gaacttatct actctcgcct gaaagaaaac ggttttatta aaaaccgcac catctctcag 2700

2760

aaatgtaaat ccccggatca atacggcgat aactgcgaag tctgcggcgc gacctacagc 2820

ccgactgaac tgatcgagcc gaaatcggtg gtttctggcg ctacgccggt aatgcgtgat 2880

tctgaacact tcttctttga tctgccctct ttcagcgaaa tgttgcaggc atggacccgc 2940

agcggtgcgt tgcaggagca ggtggcaaat aaaatgcagg agtggtttga atctggcctg 3000

caacagtggg atatctcccg cgacgcccct tacttcggtt ttgaaattcc gaacgcgccg 3060

ggcaaatatt tctacgtctg gctggacgca ccgattggct 3100

<210> 58

<211> 2650

<212> DNA

<213> Artificial sequence

<220>

<223> Lter5

<400> 58

ttgaaattcc gaacgcgccg ggcaaatatt tctacgtctg gctggacgca ccgattggct 60

acatgggttc tttcaagaat ctgtgcgaca agcgcggcga cagcgtaagc ttcgatgaat 120

actggaagaa agactccacc gccgagctgt accacttcat cggtaaagaat attgtttact 180

tccacagcct gttctggcct gccatgctgg aaggcagcaa cttccgcaag ccgtccaacc 240

tgtttgttca tggctatgtg acggtgaacg gcgcaaagat gtccaagtct cgcggcacct 300

ttattaaagc cagcacctgg ctgaatcatt ttgacgcaga cagcctgcgt tactactaca 360

ctgcgaaact ctcttcgcgc attgatgata tcgatctcaa cctggaagat ttcgttcagc 420

gtgtgaatgc cgatatcgtt aacaaagtgg ttaacctggc ctcccgtaat gcgggcttta 480

540

ccttcactga tgccgctgaa gtgattggtg aagcgtggga aagccgtgaa tttggtaaag 600

ccgtgcgcga aatcatggcg ctggctgatc tggctaaccg ctatgtcgat gaacaggctc 660

cgtgggtggt ggcgaaacag gaaggccgcg atgccgacct gcaggcaatt tgctcaatgg 720

gcatcaacct gttccgcgtg ctgatgactt acctgaagcc ggtactgccg aaactgaccg 780

agcgtgcaga agcattcctc aatacggaac tgacctggga tggtatccag caaccgctgc 840

tgggccacaa agtgaatccg ttcaaggcgc tgtataaccg catcgatatg aggcaggttg 900

aagcactggt ggaagcctct aaagaagaag taaaagccgc tgccgcgccg gtaactggcc 960

cgctggcaga tgatccgatt caggaaacca tcacctttga cgacttcgct aaagttgacc 1020

tgcgcgtggc gctgattgaa aacgcagagt ttgttgaagg ttctgacaaa ctgctgcgcc 1080

tgacgctgga tctcggcggt gaaaaacgca atgtcttctc cggtattcgt tctgcttacc 1140

cggatccgca ggcactgatt ggtcgtcaca ccattatggt ggctaacctg gcaccacgta 1200

aaatgcgctt cggtatctct gaaggcatgg tgatggctgc cggtcctggc gggaaagata 1260

ttttcctgct aagcccggat gccggtgcta aaccgggtca tcaggtgaaa taatccccct 1320

1380

ttttgtctct ctctaacccg cataaaatcg agcgtgacgt tgcgctccat ggttcctgcc 1440

ttttaatcag ttgtgatgac gcacagcgcg cagaaactcg tggcgcgtat tctgactgga 1500

tttgaacaat ccaccaagag aggtcgttgt cgtggcactg gttgcatcgc ggatgccacg 1560

1620

1680

aaagaactgc acaatgcggt taatttttga cagaccgatc accgaatctt tcgggatata 1740

ggccaccgtc gctttgccat cgatggtaac aaaatggtgt tcacaggtgc tggtcagagt 1800

gatatcgcgc acggtgacca tttcatcgac cttcattttg ttttcaatga gggtgatttt 1860

cgggaaattg gcgtaatcca gaccggagaa aatttcatcg acatacattt tagcgatgcg 1920

atgcggcgtt tccatcaaac tgtcatcagc caggtcgaga ttcagcagct gcatgatttc 1980

ggtcatatga ccagcaataa ggcttttgcg cgtttcgtta tccatttcat gcacgggcgg 2040

2100 gcgcagcggt gtttccagtc ctcgcgcaac taacgcttca

actgagtgat ggcatttatg atttctcctg caggtgtgac gcctccgccc tgcgtggggg 2160

caaagttatt aagctgattt acagcctgat tattgtgcgt gaggcggcgc acataatcca 2220

gtattcacag cgataattat tgtaattgcc gctgcctttc atcagcagat gttaaaacat 2280

cgttatgcaa atacggaagt gaaagttact cacagcacat tgaataaacg gtatgatgaa 2340

2400

ttgaaggctg gcagcaacgc tggcgacacg actccagtac cttaaactgc ccgatgacgt 2460

tcagtatctt tctccctcca cctcgtgatc acactccgcc accagtgctg tactggcttt 2520

ccggattaac ctgcaatgac gagaacttca ccaccaaggc gggtgcccag cgggtagcgg 2580

cggaactggg gattgtactg gtgatgccag acaccagccc gcgcggcgaa aaggttgcca 2640

acgacgatgg 2650

<210> 59

<211> 1298

<212> DNA

<213> Artificial sequence

<220>

<223> Fragment Cm-oriC/325k

<400> 59

tattggtaac cagaccggca ttttacgtaa tgaaccaggc atcctttctc ccacaaatat 60

cgtaagaggt tccaactttc accataatga aataagatca ctaccgggcg tattttttga 120

gttatcgaga ttttcaggag ctaaggaagc taaaatggag aaaaaaatca ctggatatac 180

caccgttgat atatcccaat ggcatcgtaa agaacatttt gaggcatttc agtcagttgc 240

tcaatgtacc tataaccaga ccgttcagct ggatattacg gcctttttaa agaccgtaaa 300

gaaaaataag cacaagtttt atccggcctt tattcacatt cttgcccgcc tgatgaatgc 360

tcatccggaa ttccgtatgg caatgaaaga cggtgagctg gtgatatggg atagtgttca 420

cccttgttac accgttttcc atgagcaaac tgaaacgttt tcatcgctct ggagtgaata 480

ccacgacgat ttccggcagt ttctacacat atattcgcaa gatgtggcgt gttacggtga 540

aaacctggcc tattcccta aagggtttat tgagaatatg tttttcgtct cagccaatcc 600

ctgggtgagt ttcaccagtt ttgatttaaa cgtggccaat atggacaact tcttcgcccc 660

cgttttcacc atgggcaaat attatacgca aggcgacaag gtgctgatgc cgctggcgat 720

tcaggttcat catgccgtct gtgatggctt ccatgtcggc agaatgctta atgaattaca 780

acagtactgc gatgagtggc agggcggggc gtaagaagat ccggcagaag aatggagtat 840

gttgtaacta aagataactt cgtataatgt atgctatacg aagttataca gatcgtgcga 900

tctactgtgg ataactctgt caggaagctt ggatcaaccg gtagttatcc aaagaacaac 960

tgttgttcag tttttgagtt gtgtataacc cctcattctg atcccagctt atacggtcca 1020

ggatcaccga tcattcacag ttaatgatcc tttccaggtt gttgatctta aaagccggat 1080

1140

ataaatagat cttcttttta atacccagga tccatttaac ataatataca ttatgcgcac 1200

ctttagttac aacatactca ggtctttctc aagccgacct agagaataaa tttatattga 1260

ttaaatgaat gtatatttca aattgatttt gtttgtta 1298

<210> 60

<211> 1298

<212> DNA

<213> Artificial sequence

<220>

<223> Fragment Cm-oriC/220k

<400> 60

caaaactgac ataaatctcc agagatgtgt tcaggagtta gaaagattat ttcttctatt 60

cgtaagaggt tccaactttc accataatga aataagatca ctaccgggcg tattttttga 120

gttatcgaga ttttcaggag ctaaggaagc taaaatggag aaaaaaatca ctggatatac 180

caccgttgat atatcccaat ggcatcgtaa agaacatttt gaggcatttc agtcagttgc 240

tcaatgtacc tataaccaga ccgttcagct ggatattacg gcctttttaa agaccgtaaa 300

gaaaaataag cacaagtttt atccggcctt tattcacatt cttgcccgcc tgatgaatgc 360

tcatccggaa ttccgtatgg caatgaaaga cggtgagctg gtgatatggg atagtgttca 420

cccttgttac accgttttcc atgagcaaac tgaaacgttt tcatcgctct ggagtgaata 480

ccacgacgat ttccggcagt ttctacacat atattcgcaa gatgtggcgt gttacggtga 540

aaacctggcc tattcccta aagggtttat tgagaatatg tttttcgtct cagccaatcc 600

ctgggtgagt ttcaccagtt ttgatttaaa cgtggccaat atggacaact tcttcgcccc 660

cgttttcacc atgggcaaat attatacgca aggcgacaag gtgctgatgc cgctggcgat 720

tcaggttcat catgccgtct gtgatggctt ccatgtcggc agaatgctta atgaattaca 780

acagtactgc gatgagtggc agggcggggc gtaagaagat ccggcagaag aatggagtat 840

gttgtaacta aagataactt cgtataatgt atgctatacg aagttataca gatcgtgcga 900

tctactgtgg ataactctgt caggaagctt ggatcaaccg gtagttatcc aaagaacaac 960

tgttgttcag tttttgagtt gtgtataacc cctcattctg atcccagctt atacggtcca 1020

ggatcaccga tcattcacag ttaatgatcc tttccaggtt gttgatctta aaagccggat 1080

1140

ataaatagat cttcttttta atacccagga tccatttaac ataatataca ttatgcgcac 1200

ctttagttac aacatactca ggtctttctc aagccgacct agaagcgatc actgctgggt 1260

tacctgtttt aacaacagcg gtatgtgggt acgcgcat 1298

<210> 61

<211> 353

<212> PRT

<213> Escherichia coli

<220>

<223> RecA

<400> 61

Met Ala Ile Asp Glu Asn Lys Gln Lys Ala Leu Ala Ala Ala Leu Gly

1 5 10 15

Gln Ile Glu Lys Gln Phe Gly Lys Gly Ser Ile Met Arg Leu Gly Glu

20 25 30

Asp Arg Ser Met Asp Val Glu Thr Ile Ser Thr Gly Ser Leu Ser Leu

35 40 45

Asp Ile Ala Leu Gly Ala Gly Gly Leu Pro Met Gly Arg Ile Val Glu

50 55 60

Ile Tyr Gly Pro Glu Ser Ser Gly Lys Thr Thr Leu Thr Leu Gln Val

65 70 75 80

Ile Ala Ala Ala Gln Arg Glu Gly Lys Thr Cys Ala Phe Ile Asp Ala

85 90 95

Glu His Ala Leu Asp Pro Ile Tyr Ala Arg Lys Leu Gly Val Asp Ile

100 105 110

Asp Asn Leu Leu Cys Ser Gln Pro Asp Thr Gly Glu Gln Ala Leu Glu

115 120 125

Ile Cys Asp Ala Leu Ala Arg Ser Gly Ala Val Asp Val Ile Val Val

130 135 140

Asp Ser Val Ala Ala Leu Thr Pro Lys Ala Glu Ile Glu Gly Glu Ile

145 150 155 160

Gly Asp Ser His Met Gly Leu Ala Ala Arg Met Met Ser Gln Ala Met

165 170 175

Arg Lys Leu Ala Gly Asn Leu Lys Gln Ser Asn Thr Leu Leu Ile Phe

180 185 190

Ile Asn Gln Ile Arg Met Lys Ile Gly Val Met Phe Gly Asn Pro Glu

195 200 205

Thr Thr Thr Gly Gly Asn Ala Leu Lys Phe Tyr Ala Ser Val Arg Leu

210 215 220

Asp Ile Arg Arg Ile Gly Ala Val Lys Glu Gly Glu Asn Val Val Gly

225 230 235 240

Ser Glu Thr Arg Val Lys Val Val Lys Asn Lys Ile Ala Ala Pro Phe

245 250 255

Lys Gln Ala Glu Phe Gln Ile Leu Tyr Gly Glu Gly Ile Asn Phe Tyr

260 265 270

Gly Glu Leu Val Asp Leu Gly Val Lys Glu Lys Leu Ile Glu Lys Ala

275 280 285

Gly Ala Trp Tyr Ser Tyr Lys Gly Glu Lys Ile Gly Gln Gly Lys Ala

290 295 300

Asn Ala Thr Ala Trp Leu Lys Asp Asn Pro Glu Thr Ala Lys Glu Ile

305 310 315 320

Glu Lys Lys Val Arg Glu Leu Leu Leu Ser Asn Pro Asn Ser Thr Pro

325 330 335

Asp Phe Ser Val Asp Asp Ser Glu Gly Val Ala Glu Thr Asn Glu Asp

340 345 350

Phe

<210> 62

<211> 1509

<212> DNA

<213> Artificial sequence

<220>

<223> Km-oriC (DCW49)

<400> 62

agtcacatct taacggtgaa gctgaagtag aaaaacgtgt tacagcatca gttggctcgt 60

gtcggcttga gaaagacctg agtatgttgt aactaaaggt gcgcataatg tatattatgt 120

taaatggatc ctgggtatta aaaagaagat ctatttattt agagatctgt tctattgtga 180

240

cctggaaagg atcattaact gtgaatgatc ggtgatcctg gaccgtataa gctgggatca 300

gaatgagggg ttatacacaa ctcaaaaact gaacaacagt tgttctttgg ataactaccg 360

gttgatccaa gcttcctgac agagttatcc acagtagatc gcacgatctg tataacttcg 420

tatagcatac attatacgaa gttatcttta gttacaacat actccattct tctgccggat 480

cttcgggaaa gccacgttgt gtctcaaaat ctctgatgtt acattgcaca agataaaaat 540

atatcatcat gaacaataaa actgtctgct tacataaaca gtaatacaag gggtgttatg 600

agccatattc aacgggaaac gtcttgctcg aggccgcgat taaattccaa catggatgct 660

gatttatatg ggtataaatg ggctcgcgat aatgtcgggc aatcaggtgc gacaatctat 720

cgattgtatg ggaagcccga tgcgccagag ttgtttctga aacatggcaa aggtagcgtt 780

gccaatgatg ttacagatga gatggtcaga ctaaactggc tgacggaatt tatgcctctt 840

ccgaccatca agcattttat ccgtactcct gatgatgcat ggttactcac cactgcgatc 900

cccgggaaaa cagcattcca ggtattagaa gaatatcctg attcaggtga aaatattgtt 960

gatgcgctgg cagtgttcct gcgccggttg cattcgattc ctgtttgtaa ttgtcctttt 1020

1080

gatgcgagtg attttgatga cgagcgtaat ggctggcctg ttgaacaagt ctggaaagaa 1140

atgcataagc ttttgccatt ctcaccggat tcagtcgtca ctcatggtga tttctcactt 1200

1260

atcgcagacc gatacgga tcttgccatc ctatggaact gcctcggtga gttttctcct 1320

1380

cagtttcatt tgatgctcga tgagtttttc taatcagaat tggttaattg gttgtaacac 1440

tggcagagct tctaaaaccg tgactgcgga tatcccgatt gtgggggatg ctcgccaggt 1500

cctcgaaca 1509

<210> 63

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 63

ctatgcggca tcagagcag 19

<210> 64

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 64

gttaagccag ccccgacac 19

<210> 65

<211> 500

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 65

cgcgtcagcg ggtgttggcg ggtgtcgggg ctggcttaac agtactgcga tgagtggcag 60

ggcggggcgt aattttttta aggcagttat tggtgccctt aaacgcctgg ttgctacgcc 120

tgaataagtg ataataagcg gatgaatggc agaaattcga tgataagctg tcaaacatga 180

gaattggtcg acggcgcgcc aaagcttgca tgcctgcagc cgcgtaacct ggcaaaatcg 240

gttacggttg agtaataaat ggatgccctg cgtaagcggg gcacatttca ttacctcttt 300

ctccgcaccc gacatagata ataacttcgt atagtataca ttatacgaag ttatctagta 360

gacttaatta aggatcgatc cggcgcgcca atagtcatgc cccgcgccca cgggaaggag 420

ctgactgggt tgaaggctct caagggcatc ggtcgagctt ctatgcggca tcagagcaga 480

ttgtactgag agtgcaccat 500

<210> 66

<211> 60

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 66

tgcgtaagcg gggcacattt cattacctct ttctccgcac gctctgccag tgttacaacc 60

<210> 67

<211> 60

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 67

taatgtatac tatacgaagt tattatctat gtcgggtgct aacgcggtat gaaaatggat 60

<---

Claims (27)

1. A method for obtaining DNA, where the specified method includes: obtaining a reaction solution containing fragments of linear double-stranded DNA of two or more types, a protein having recombinase activity of the RecA family, and a 3' → 5'-exonuclease specific for linear double-stranded DNA, and obtaining linear or circular DNA in a reaction solution by linking two or more types of DNA fragments to each other in regions having homologous base sequences, or in regions having complementary base sequences, in one reaction, where regions having homologous base sequences or regions having complementary base sequences are present at or near the end of the DNA fragment, where regions having homologous base sequences or regions having complementary base sequences are 20 bp long. or more where the binding reaction of DNA fragments of two or more types is carried out at a temperature in the range of 25-48°C, and where the reaction solution contains a regenerating enzyme for nucleoside triphosphates or deoxynucleotide triphosphates and its substrate. 2. The method for producing DNA according to claim 1, wherein the reaction solution additionally contains a 3'→5'-exonuclease specific for single-stranded DNA. 3. The method of obtaining DNA according to any one of paragraphs. 1 and 2, where the combination of the regenerating enzyme and its substrate is at least one combination selected from the group consisting of a combination of creatine kinase and creatine phosphate, a combination of pyruvate kinase and phosphoenol pyruvate, a combination of acetate kinase and acetyl phosphate, a combination of polyphosphate kinase and polyphosphate and a combination of nucleoside diphosphate kinase and nucleoside triphosphate. 4. The method of obtaining DNA according to any one of paragraphs. 1-3, where linear or circular DNA is obtained by linking 7 or more DNA fragments. 5. The method of obtaining DNA according to any one of paragraphs. 1-4, where the reaction solution contains one or more substances selected from the group consisting of tetramethylammonium chloride and dimethylsulfoxide. 6. The method of obtaining DNA according to any one of paragraphs. 1-5, where the protein with recombinase activity of the RecA family is uvsX, and the reaction solution additionally contains uvsY. 7. The method of obtaining DNA according to claim 6, where the region having homologous base sequences have a length of 20 p. up to 500 bp 8. The method of obtaining DNA according to any one of paragraphs. 1-7, where the reaction solution at the beginning of the binding reaction of DNA fragments of two or more types contains DNA fragments of two or more types in the same molar concentration. 9. The method of obtaining DNA according to any one of paragraphs. 1-8, further comprising repairing gaps and nicks in the resulting linear or circular DNA using gap repair enzymes. 10. The method for producing DNA according to claim 9, further comprising heat treatment of the obtained linear or circular DNA at 50-70°C, followed by rapid cooling to a temperature of 10°C or less, and then gap and nick repair using gap repair enzymes. 11. The method of obtaining DNA according to any one of paragraphs. 1-8, where the DNA obtained by binding is linear, and PCR is carried out directly using linear DNA as a template. 12. The method of obtaining DNA according to any one of paragraphs. 1-8, where the DNA obtained by binding is a circular DNA containing an origin of replication sequence capable of binding to an enzyme having DnaA activity, and form a reaction mixture containing cDNA, the first group of enzymes that catalyze the replication of cDNA; the second group of enzymes that catalyze the binding reaction of Okazaki fragments and synthesize two sister circular DNA, consisting of catenan; a third group of enzymes that catalyze the separation of two sister circular DNAs, and dNTPs. 13. The method of obtaining DNA according to any one of paragraphs. 1-8, further comprising introducing the resulting linear or circular DNA into the microorganism and amplifying double-stranded DNA with gap and nick repair. 14. Kit for binding DNA fragments, containing: a protein with recombinase activity of the RecA family, a 3'→5'-exonuclease specific for linear double-stranded DNA, and a regenerating enzyme for nucleoside triphosphates or deoxynucleotide triphosphates and its substrate, where the specified set is used to obtain linear or circular DNA by linking two or more types of linear double-stranded DNA fragments to each other in areas having homologous base sequences, or areas having complementary base sequences, where regions having homologous base sequences or regions having complementary base sequences are present at or near the end of the DNA fragment, and where regions having homologous base sequences or regions having complementary base sequences are 20 bp long. or more. 15. A kit for binding DNA fragments according to claim 14, additionally containing a 3' → 5'-exonuclease specific for single-stranded DNA. 16. A kit for binding DNA fragments according to claim 14 or 15, where the combination of the regenerating enzyme and its substrate is at least one combination selected from the group consisting of a combination of creatine kinase and creatine phosphate, a combination of pyruvate kinase and phosphoenol pyruvate, combinations acetate kinase and acetyl phosphate, combinations of polyphosphate kinase and polyphosphate, and combinations of nucleoside diphosphate kinase and nucleoside triphosphate.

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